Interferon Production Using Short RNA Duplexes

ABSTRACT

The present invention provides a small hairpin nucleic acid molecule that is capable of stimulating interferon production. The nucleic acid molecule of the present invention has a double-stranded section of less than 19 base pairs and at least one blunt end. In certain embodiments, the molecule comprises a 5′ triphosphate or a 5′ diphosphate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of, and claims priority to, U.S.application Ser. No. 14/776,463, filed Sep. 14, 2015, now allowed, whichis the U.S. national stage application filed under 35 U.S.C. § 371claiming benefit to International Patent Application No. PCT/US14/25578,filed Mar. 13, 2014, which claims priority under 35 U.S.C. § 119(e) toU.S. Provisional Application No. 61/779,514, filed Mar. 13, 2013, eachof which is hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under RO1 AI089826-03awarded by the National Institutes of Health (NIH). The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Retinoic acid-inducible gene 1 (RIG-1), melanomadifferentiation-associated gene 5 (MDA5) and laboratory of genetics andphysiology 2(LGP2), comprise the RIG-I like receptor (RLR) class ofintracellular pattern recognition receptors (PRRs) that defend againstbacterial and viral infection by recognizing foreign RNAs in thecytoplasm and eliciting an innate immune response through the productionof pro-inflammatory cytokines and type I interferons (Abdullah et al.,2012, EMBO J 31:4153-4164; Kato et al., 2011, Immunol Rev 243:91-98;Ramos and Gale, 2011, Curr Opin Tirol 1:167-176). RIG-I recognizes bothself and non-self RNA, including positive and negative stranded RNAviruses, RNA fragments produced by RNA Polymerase III either from DNAviruses like the Epstein-Barr virus or AT-rich double stranded DNAtemplates (Ablasser et al., 2009, Nat Immunol 10:1065-1072; Chiu et al.,2009, Cell 138:576-591), RNA cleavage products of the antiviralendoribonuclease RNAse L (Malathi et al., 2007, Nature 448:816-819;Malathi et al., 2010, RNA 16: 2108-2119), synthetic poly I:C (Kato etal., 2008, J Exp Med 205:1601-1610), and even RNA aptamers lacking a5′triphosphate (Hwang et al., 2012, Nucleic Acids Res 40(6):2724-33). Ofthese substrates, the simplest RNA molecule commonly reported toactivate the RIG-I signaling pathway is 5′ triphosphorylated,blunt-ended 19-mer duplex RNA (Schlee et al., 2009, Immunity 31:25-34;Schmidt et al., 2009, Proc Natl Acad Sci USA 106:12067-12072). Moreover,RIG-I exhibits a strong preference for 5′triphosphorylated blunt ends ofduplex RNA, and will tolerate 3′ but not 5′overhangs (Schlee et al.,2009, Immunity 31:25-34). RIG-I's distinct pathogen associated molecularpattern (PAMP) is therefore defined as duplex RNA containing a5′triphosphate moiety, although only duplex RNA appears to be absolutelyrequired for RIG-I recognition (Lu et al., 2011, Nucleic Acids Res39:1565-1575).

RLRs are part of a larger group of duplex RNA activated. ATPases (DRAs)that also includes Dicer and Dicer-Related Helicases (DRFIs) (Luo etal., 2012a, RNA Biol. 2012 Dec. 10; 10(1)). Besides recognizing duplexRNA, these helicases share the common characteristic that they do notfunction as conventional helicases (i.e., they do not catalyze strandseparation) (Luo et al., 2012, RNA Biol. 2012 Dec. 10; 10(1); Pyle,2008, Annu Rev Biophys 37:317-336). All DRAs share a common superfamily2 helicase core comprised of two RecA-like domains, HEL1 and HEL2, and aconserved insertion domain in HEL2, Hel2i. Except Dicer, DRAs contain aconserved C-terminal domain (CTD), responsible for modulating thefunction of each helicase and imparting substrate RNA specificity. InRIG-I, the CTD provides this specificity by recognizing 5′triphosphates(Lu et al., 2010, Structure 18:1032-1043; Wang et al., 2010, Nat StructMol Biol 17:781-787). Initially, the RIG-I CTD was incorrectly annotatedas a repressor domain (Saito et al., 2007, Proc. Natl Acad Sci USA104:582-587), however mutant RIG-I constructs lacking a. CTD are unableto stimulate an interferon response (Kageyama et al., 2011, BiochemBiophys Res Commun 415:75-81), suggesting a role for the CTD beyondautorepression.

RIG-I and MDA5 are unique among DRAs because they contain tandem caspaseactivation and recruitment domains (CARDs) at their N-termini thatundergo ubiquitination upon substrate binding and subsequently initiatedownstream signaling by interacting with the CARD domain of themitochondrial adaptor protein MAVS (Jiang et al., 2012, Immunity36:959-973). LGP2 lacks the N-terminal CARD domains, but is implicatedin the regulation of the innate immune response as a modulator of RIG-Iand MDA5 activity (Bamming and Horvath, 2009, J Biol Chem 284:9700-9712;Jiang et al., 2012, Immunity 36:959-973; Satoh et al., 2010, Proc NatlAcad Sci USA 107:1512-1517). RIG-1 is normally found in the cytoplasm inan auto-repressed conformation, with the tandem CARDs partially occludedby an interaction with the Hel2i domain (Kowalinski et al., 2011, Cell147:423-435). Binding to an RNA substrate produces a ternary complexcompetent for ATP binding and hydrolysis, exposing the CARD domains,although the precise role of ATP binding and hydrolysis in displacingthe CARDs is still unclear. A comprehensive mutational analysis ofRIG-I, MIDAS, and LGP2 yielded several conventional Motif I-V mutantslacking catalytic activity, but found no correlation between ATPhydrolysis and IFN-β response (Bamming and Horvath, 2009, J Biol Chem284:9700-9712). It has recently been proposed that ATP binding isrequired for signaling based on a RIG-I structural analysis (Luo et al.,2012b, Structure 20:1983-1988), and this is further supported by theobservation that mutations in motif I, an ATP binding motif, disruptRIG-I-dependent response (Bamming and Horvath, 2009, J Biol Chem284:9700-9712).

Structural studies of mouse, human, and duck RIG-I truncations haveenhanced the understanding of how RIG-I recognizes RNA and utilizes ATP(Civril et al., 2011, EMBO Rep 12:1127-1134; Jiang et al., 2011, Nature479:423-427; Kowalinski et al., 2011, Cell 147:423-435; Luo et al.,2011, Cell 147:409-422). Unfortunately, in the only RIG-I structure withthe CARD domains present, the protein is in an inactive, apo-state, andlacks the CTD. This leaves several important questions unansweredregarding the role of both RNA and ATP in RIG-I's innate immuneresponse, and the relative positions of the CTD and CARDs in the activeRIG-I conformation. Intriguingly, in all of the RIG-I:RNA complexstructures, the RIG-I CTD caps the 5′ end of the RNA, regardless of thelength of the bound duplex. RIG-I's preference for the end of the duplexRNA in these structures is also independent of a 5′triphosphate.Furthermore, the RIG-I helicase domain exhibits a weak affinity for both5′OH and 5′ppp duplex RNA, with a K_(D) in the micromolar range (Jianget al., 2011, Nature 479:423-427; Vela et al., 2012, J Biol Chem287:42564-42573), suggesting that internal duplex stem binding may playa lesser role in RIG-I stimulation.

Several studies have reported the RNA-induced multimerization of RIG-Iusing a variety of techniques, including size exclusion chromatography,atomic force microscopy (AFM), and electrophoretic mobility shift assay(EMSA) experiments (Beckham et al., 2013, Nucleic Acids Res. 2013 Jan.15; Binder et al., 2011, J Biol Chem 286(31):27278-87; Feng et al.,2012, Protein Cell. 2012 Dec. 20; Schmidt et al., 2009, Proc Natl AcadSci USA 106:12067-12072). This oligomerization might occur viainteractions between two or more RIG-I molecules bound to the same RNAsubstrate, or through protein-protein interactions between independentternary complexes subsequent to RNA stimulation, or conceivably throughsome combination of these two scenarios. An IRF3 dimerization assayreconstituted in vitro demonstrated that poly-ubiquitin chains inducethe formation of a RIG-I tetramer composed of four RIG-I:RNA units andfour poly-ubiquitin chains (Jiang et al., 2012, Immunity 36:959-973).Whereas MDA5 forms long cooperative filaments on RNA with distinctprotein-protein contacts required for activation and consequentlyprefers longer RNA substrates than RIG-I (Berke and Modis, 2012, EMBO J31:1714-1726; Berke et al., 2012, Proc Natl Acad Sci USA109:18437-18441; Jiang et al., 2012, Immunity 36:959-973; Peisley etal., 2011, Proc Natl Acad Sci USA 108: 21010-21015), the oligomerizationstate required for RIG-I activation and RIG-I's preference for smallersubstrates is not well understood (Kolakofsky et al., 2012, RNA18:2118-2127).

There still remains a need in the art for compositions and method tostudy the role of RIG-I and means of regulating RIG-I. The presentinvention satisfies this need in the art.

SUMMARY OF THE INVENTION

The present invention provides a composition comprising a nucleic acidcapable of inducing interferon production. The molecule comprises adouble-stranded section of less than 19 base pairs and at least oneblunt end. In one embodiment, the nucleic acid molecule comprises asingle strand nucleic acid molecule which forms a hairpin structurecomprising the double-stranded section and a loop. In one embodiment,the nucleic acid molecule comprises a double-stranded nucleic acidmolecule and two blunt ends. In one embodiment, the nucleic acidmolecule comprises at least one of the group consisting of a 5′triphosphate and a 5′ diphosphate. In one embodiment, the molecule iscapable of entering the nucleus.

In one embodiment, the molecule comprises a modified phosphodiesterbackbone. In one embodiment, the molecule comprises at least one2′-modified nucleotide. In one embodiment, the 2′-modified nucleotidecomprises a modification selected from the group consisting of:2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), and 2′-O—N-methylacetamido (2′-O-NMA). In one embodiment,the molecule comprises at least one modified phosphate group. In oneembodiment, the molecule comprises at least one modified base. In oneembodiment, the double-stranded section comprises one or more mispairedbases.

The present invention provides a method for inducing type I interferonproduction in a cell. The method comprises contacting the cell with anucleic acid molecule, wherein the molecule comprises a double-strandedsection of less than 19 base pairs and at least one blunt end. In oneembodiment, the nucleic acid molecule comprises a single strand nucleicacid molecule which forms a hairpin structure comprising thedouble-stranded section and a loop. In one embodiment, the nucleic acidmolecule comprises a double-stranded nucleic acid molecule and two bluntends. In one embodiment, the nucleic acid molecule comprises at leastone of the group consisting of a 5′ triphosphate and a 5′ diphosphate.In one embodiment, the molecule is capable of entering the nucleus.

In one embodiment, the molecule comprises a modified phosphodiesterbackbone. In one embodiment, the molecule comprises at least one2′-modified nucleotide. In one embodiment, the 2′-modified nucleotidecomprises a modification selected from the group consisting of:2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), and 2′-O—N-methylacetamido (2′-O-NMA). In one embodiment,the molecule comprises at least one modified phosphate group. In oneembodiment, the molecule comprises at least one modified base. In oneembodiment, the double-stranded section comprises one or more mispairedbases.

The present invention provides a method for treating a disease ordisorder in a subject in need thereof by inducing type I interferonproduction in a cell of the subject. The method comprises contacting thecell with a nucleic acid molecule, wherein the molecule comprises adouble-stranded section of less than 19 base pairs and at least oneblunt end. In one embodiment, the nucleic acid molecule comprises asingle strand nucleic acid molecule which forms a hairpin structurecomprising the double-stranded section and a loop. In one embodiment,the nucleic acid molecule comprises a double-stranded nucleic acidmolecule and two blunt ends. In one embodiment, the nucleic acidmolecule comprises at least one of the group consisting of a 5′triphosphate and a 5′ diphosphate. In one embodiment, the molecule iscapable of entering the nucleus.

In one embodiment, the molecule comprises a modified phosphodiesterbackbone. In one embodiment, the molecule comprises at least one2′-modified nucleotide. In one embodiment, the 2′-modified nucleotidecomprises a modification selected from the group consisting of:2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), and 2′-O—N-methylacetamido (2′-O-NMA). In one embodiment,the molecule comprises at least one modified phosphate group. In oneembodiment, the molecule comprises at least one modified base. In oneembodiment, the double-stranded section comprises one or more mispairedbases.

In one embodiment, the disease or disorder is selected from the groupconsisting of a bacterial infection, a viral infection, a parasiticinfection, cancer, an autoimmune disease, an inflammatory disorder, anda respiratory disorder.

The present invention provides a pharmaceutical composition comprising anucleic acid molecule capable of inducing interferon production and apharmaceutically acceptable carrier, wherein the molecule comprises adouble-stranded section of less than 19 base pairs and at least oneblunt end. In one embodiment, the nucleic acid molecule comprises asingle strand nucleic acid molecule which forms a hairpin structurecomprising the double-stranded section and a loop. In one embodiment,the nucleic acid molecule comprises a double-stranded nucleic acidmolecule and two blunt ends. In one embodiment, the nucleic acidmolecule comprises at least one of the group consisting of a 5′triphosphate and a 5′ diphosphate. In one embodiment, the pharmaceuticalcomposition further comprises at least one agent selected from animmunostimulatory agent, an antigen, an anti-viral agent, ananti-bacterial agent, an anti-tumor agent, retinoic acid, IFN-α, andIFN-β.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIGS. 1A-1E depict the results of structural analysis demonstrating thatthe HEL2i domain scans along the duplex RNA backbone. Three distinctconformations of RIG-I (ΔCARDs: 1-229):GC10 with an empty ATP-bindingpocket (pdb:3zd6), and in complex with a SO₄ ²⁻ (pdb:2ykg) and ADP-Mg²⁺(pdb:3zd7). GC10 is a palindromic RNA duplex of repeating ‘GC’ with a5′hydroxyl. FIG. 1A depicts the alignment of the three conformations.FIG. 1B depicts the interface between the HEL2i and the duplex RNA. Keyresidues in the HEL2i domain, Q511 and K508, are involved in RNA bindingand are shown as sticks. Using residue E530 as the reference, there is a14 Å movement of the HEL2i domain between conformations 1 and 2, and a16 Å movement between conformations 2 and 3. FIG. 1C is a close-up viewof the pincer domain, highlighting the motions of pincer1 (the firstα-helix). The change in the angle between pincer1 and pincer2 (thesecond α-helix) is 11°. FIG. 1D depicts the ATP-binding pocket of thesuperimposed structures. Ligands (SO₄ ²⁻ and ADP-Mg²⁺) and the keyresidues (K270 from motif I; D372 and E373 from motif II) are shown assticks. FIG. 1E is a diagram of the RIG-I:RNA duplex interface. Theclosest distances between the RNA and the residues K508 and Q511 fromthe HEL2i domain are highlighted and shown as dashed lines. CARDs,caspase activation and recruitment domains; RIG-I, retinoicacid-inducible gene-I.

FIG. 2 is a set of graphs depicting the results of an experimentdemonstrating that RIG-I binds hairpins with one triphosphate with a 1:1stoichiometry. Hydrodynamic analysis of RIG-I in complex with 5′ppp10L,5′ppp20L, 5′ppp30L and 5′pppGC22 RNA. c(s) distributions for each SVexperiment were plotted against the sedimentation coefficient(s_(20,w)). Peak s_(20,w) values for each distribution are 6.0 for RIG-Ialone and 6.2, 6.4, 6.9 and 9.3 for RIG-I: 5′ppp10L, 5′ppp20L, 5′ppp30Land 5′pppGC22 complexes, respectively. Estimated molecular weights fromSedfit are 106 kDa (ƒ/ƒ₀=1.31), 113 kDa (ƒ/ƒ₀=1.31), 121 kDa (86=1.53),133 kDa (ƒ/ƒ₀=1.57) and 228 kDa (86=1.45), respectively. Models of RIG-Ibound to each RNA construct are shown next to each c(s) distribution.RIG-I, retinoic acid-inducible gene-I; SV, sedimentation velocity.

FIGS. 3A-3D depict the results of experiments demonstrating that RIG-Iis stimulated by the ends of poly I:C. (FIG. 3A) LMW poly I:C wasfractionated on an analytical Superdex 200 size exclusion chromatographycolumn and separated into seven fractions on a 15% polyacrylamide, 4Murea semi-denaturing gel stained with ethidium bromide (marker is inbase pairs). (FIG. 3B) ATPase activity of RIG-I stimulated by 0-15 ng/μlof the poly I:C fractions A1, A3, A5 and A7 at 5 mM ATP. The data werefit to the quadratic form of the Briggs-Haldane equation with theassumption that the k_(cat) values are the same for all the fractions.(FIG. 3C) The K_(m,ATP) for RIG-I stimulated by 15 ng/μl of the poly I:Cfractions A1-A7 while varying the ATP concentration from 0-5 mM ATP.(FIG. 3D) The calculated K_(m,RNA) for RIG-I stimulated by 0-15 ng/μl ofthe poly I:C fractions A1-A7 at 5 mM ATP. The K_(m) values in the leftpanel are in ng/μl and values in the right panel are in nM for all sevenfractions on the basis of the estimated sizes of each fraction. Errorbars for the poly I:C data report the standard deviation across sixexperiments. LMW, low molecular weight; RIG-I, retinoic acid-induciblegene-I.

FIGS. 4A-4D depict the results of experiments demonstrating the in vitroand cell culture activities of RIG-I in response to short duplex RNAs.(FIG. 4A) The K_(m,ATP) of RIG-I stimulated by a library of duplex RNAconstructs at ATP concentrations varying between 0 and 5 mM. (FIG. 4B)The K_(m,RNA) of RIG-I stimulated by a library of duplex RNA constructsat RNA concentrations varying between 0 and 500 nM. The K_(m,RNA) forthe GC8 duplex is −100 nM and did not fit on the scale. (FIG. 4C) Thek_(cat) summary averaged from the K_(m,ATP) and K_(m,RNA) experiments.(FIG. 4D) RIG-I stimulated IFN-0 production was measured in 293T cells.RIG-I was stimulated by 5′triphosphorylated hairpins (20-650 nM) and thepositive controls, poly I:C (15-500 ng/well) and 5′pppGC22 (20-650 nM).The increase in RNA concentration is indicated by a darkening colorgradient. The relative luciferase is the firefly luciferase (IFN-βreporter) divided by the constitutively expressed Renilla luciferase.Error bars for the ATPase data report the standard deviation from atleast three measurements. Error bars for the cell culture data reportthe standard error of the mean from three measurements. IFN-β,interferon-β; RIG-I, retinoic acid-inducible gene-I. The constructs andnucleic acid sequences of the constructs used are listed in Table 2.

FIGS. 5A-5B are a schematic model of RIG-I activation. (1) RNA bindingis the first trigger of the RIG-I-mediated interferon response. The CTDbinds firmly to the 5′ end of the duplex RNA. The CARD domains rest onthe HEL2i domain (Kowalinski et al., 2011, Cell, 147: 423-435) andlikely are not displaced upon RNA binding. (2) ATP binding serves as thesecond trigger, whereupon HEL1 and HEL2 close and HEL2 initiatescontacts with the tracking strand, creating a clash between the CTD andthe CARDs (Luo et al., 2012a, RNA Biol, 10: 111-120). HEL2i scanningmight be directly linked to ATP binding and hydrolysis, or it might movestochastically. (3) Once the CARD domains are released, a 1:1:1RIG-I:RNA:ATP ternary complex is competent for signalling and activationof MAVS. (4) Ubiquitin-mediated multimerization (tetraubiquitin shown inorange) of RIG-I through the CARD domains might be required for MAVSactivation (Jiang et al., 2012, Immunity, 36: 959-973; Gack et al.,2007, Nature, 446: 916-920). CARD, caspase activation and recruitmentdomain; CTD, carboxy-terminal domain; MAVS, mitochondrialantiviral-signalling protein; RIG-I, retinoic acid-inducible gene-I.

FIGS. 6A-6B depict the results of K_(m,ATP) and K_(m,RNA) ATPaseexperiments on LMW poly I:C. FIG. 6A depicts the ATPase activity ofRIG-I stimulated by 500 ng/μL LMW poly I:C while varying the ATPconcentration from 0 to 5 mM ATP. Error bars report the standarddeviation from 4 experiments. FIG. 6B depicts the ATPase activity ofRIG-I at 5 mM ATP while varying LMW poly I:C from 0 to 500 ng/μL. Errorbars report the standard deviation from 4 experiments. The averagek_(cat) from both experiments was 4.9 s⁻¹, and the K_(m,ATP) wasapproximately 700 μM. The K_(m,RNA) was 2.4 ng/μL, which is difficult tointerpret because it cannot be expressed as a nanomolar value due to theheterogeneity of poly I:C samples

FIGS. 7A-7C depict the results of K_(m,ATP) and K_(m,RNA) ATPaseexperiments on short duplex RNA. ATPase activity of RIG-I stimulated byvarious length RNAs including 5′ triphosphorylated hairpins, 5′hydroxylduplexes, and 5′ triphosphorylated duplexes. The K_(m,ATP) of RIG-I (10nM enzyme) stimulated by each RNA was measured by varying the ATPconcentrations ranging from 0 to 5 mM at 500 nM RNA. The K_(m,RNA) ofRIG-I (5 nM enzyme) stimulated by each RNA was measured by varying theRNA concentrations ranging from 0 to 500 nM at 5 mM ATP. A small basalactivity (0 nM RNA) is measured for RIG-I of less than 1 per second.Error bars for the K_(m,ATP) and K_(m,RNA) experiments report thestandard error of the mean from 4 experiments. The last column of graphsplots the average k_(cat) values calculated from Briggs-Haldane fitsfrom both the K_(m,ATP) and K_(m,RNA) experiments. Error bars for thek_(cat) summary report the standard deviation measured across 6experiments, in which each experiment was comprised of an averagedduplicate dataset for each RNA or ATP concentration. (FIG. 7A) ATPasemeasurements on 4 triphosphorylated hairpins with a duplex region of 8,10, 20, and 30 nucleotides with a UUCG hairpin. (FIG. 7B) ATPasemeasurements on 6 double stranded RNA duplexes with 5′hydroxyl of length8, 10, 12, 14, 18, and 22. (FIG. 7C) ATPase measurements on 4 doublestranded RNA duplexes with a 5′triphosphate of length 10, 12, and 22.Table 2 lists the RNA sequences used in this study. Similar k_(cat)values were observed for RIG-I stimulated by the 5′ppp8L hairpin andGCB. However, in the case of the hairpin, a 5.2 nM K_(m,RNA) wasobserved, approximately 20-fold smaller than GCB, perhaps because5′ppp8L contains a ‘UUCG’ tetraloop, which may accommodate the HEL2iflexibility seen in the crystal structures.

FIG. 8 depicts the result of an experiment using a mock control forHEK293T cell culture IFN production. The IFN-β production in 293T cellsoverexpressing RIG-I (and a mock control not overexpressing RIG-I) wasmeasured in the absence (left) and presence (right) of poly I:Cstimulation. The relative luciferase is the firefly luciferase (IFN-βreporter) divided by the Renilla luciferase. The following protocol wasadapted from Luo et. al. (Luo et al, 2011). 293T cells were seeded at−50,000 cells per well in 24 well plates. The next day, 293T cells weretransfected with 30 ng of pRLTK, 178 ng of a firefly IFN-β reporter, and3 ng (or none for mock) of pUNO-RIG-I per well using lipofectin(Invitrogen). After 24 hours, 293T cells were transfected with 1 μg ofpoly I:C (or none for negative control) using mRNA transfection reagent(MIRUS). After 16 hours, cells were harvested and assayed for fireflyand Renilla luciferase using the Promega Dual Luciferase Reporter assaysystem. Error bars report the standard deviation from 6 experiments forunstimulated and 12 experiments for stimulated.

FIG. 9, depicts the results of an experiment examining the cell cultureIFN production on different lengths of poly I:C. The IFN-β responses tothe fractions of poly I:C was measured in HEK 293T cells transfectedwith pUNO-RIG-I, an IFN-β/Firefly luciferase reporter, and a pRL-TKreporter (note that the poly I:C data is the same as in FIG. 4D and wasdone side by side with Fraction A1-A7 shown here). The charts displaythe measured relative luciferase ratio of Firefly luminescence overRenilla luminescence from 293T cells in which RIG-I was stimulated bythe fractions of poly I:C, and also a single stranded poly U and no RNA(serum free media) control. The range of RNA concentrations spansbetween 31 to 250 ng per well displayed in the figure by a darkeningcolor gradient from low to high RNA concentration. Error bars report thestandard error of the mean from 3 measurements.

FIGS. 10A-10C are a series of images showing ATPase activity of 5, 10,25, and 50 nM RIG-I stimulated by hairpin and duplex RNA at ATPconcentrations ranging from 0 to 5 mM. Measurements are reported as ATPmolecules hydrolyzed per second. FIG. 10A demonstrates the results ofvarying concentrations of RIG-I stimulated by 1 μM of 5′ppp10L. Errorbars report SEM from 4 experiments at each ATP concentration. FIG. 10Bshows the results of varying concentrations of RIG-I stimulated by 1 μMof 5′pppGC22. Error bars report SEM from 4 experiments at each ATPconcentration. FIG. 10C is a graph showing the k_(cat) values from thefit to the hyperbolic form of the Briggs-Haldane equation are plotted ateach enzyme concentration for 5′ppp10L and 5′pppGC22. Error bars reportthe standard error from the fit.

FIG. 11 shows IFN-β stimulation by 5′OH palindromic duplexes. The IFN-βresponses to 5′OH palindromic ‘GC’ duplexes was measured in HEK 293Tcells transfected with pUNO-RIG-I, an IFN-β/Firefly luciferase reporter,and a pRL-TK reporter. The charts display the measured relativeluciferase ratio of Firefly luminescence over Renilla luminescence from293T cells in which RIG-I was stimulated by 5′OH ‘GC’ palindromicduplexes of length of 8, 10, 12, 14, 18, and 22. The range ofconcentrations for each RNA spans between 20 to 650 nM and are displayedin the figure by a darkening color gradient from low to high RNAconcentration. Error bars report the standard error of the mean from 3measurements.

FIG. 12 is a graph depicting the results of a representative experimentdepicting serum Interferon alpha levels after treatment with shorthairpin RNAs: Mice were injected in the tail vein with jetPEI/RNAcomplex (i.v.), and serum was collected at 5 hours post-injection. Thedose used per mouse was as follows: polyIC=25 ug, hp10=640 uM (25.15ug), hp414=640 uM (33.4 ug). Four mice were used for each condition. Theresults indicate that very high levels of IFNalpha are induced by shRNAsand polyIC, and not by the vehicle control. Notably, the shRNAs inducemore IFNalpha than polyIC. Note that hp10 is a 5′-triphosphorylated 10base-pair duplex with a UUCG tetraloop at one end (5′ppp10L from FIGS.4A-4D and FIGS. 7A-7C) and hp14 is a 5′-triphosphorylated 14 base pairduplex with a UUCG tetraloop at one end. The polyIC is low molecularweight poly IC.

FIG. 13 is a graph depicting the results of a representative experimentdepicting serum Interferon alpha levels after treatment withdephosphorylated and triphosphorylated short hairpin RNAs:Mice wereinjected in the tail vein with jetPEI/RNA complex (i.v.), and serum wascollected at 5 hours post-injection, n=3 per group. RNA #1 (center,ppp10L+enz)=5′ppp10L transcribed and then treated with Dnase/Prot K,then phenol extraction and ethanol precipitation. RNA #2 (left, OH10L+enz)=5′OH10L, which is transcribed 5′ppp10L treated with CIP, thenenzyme treated/purified as above. RNA #3 (right, ppp10L synth)=5′ppp10Lthat is machine-synthesized, abiological. It is demonstrated that only5′ppp10L (whether transcribed or synthetic), and not RNA lackingtriphosphate (left), induces interferon. Both transcribed andsynthesized 5′ppp10L induce IFN to a similar degree, although thesynthetic triphosphorylated RNA is slightly more active. Extra enzymetreatment and purification of transcribed 5′ppp10L does not impact IFNlevels.

DETAILED DESCRIPTION

The present invention provides a nucleic acid molecule that can activatethe interferon response of one or more pattern recognition receptors(PRRs). The invention is based on the identification of a minimal RNAsubstrate to which RIG-I binds whereby the substrate stimulates theATPase activity by RIG-I and elicits an interferon response in vivo.Accordingly, the invention provides compositions and methods forinducing the interferon response of one or more PRRs. For example, thecompositions and methods described herein may activate any PRRincluding, but not limited to, the RIG-I like receptor (RLR) class ofPRRs, which include RIG-I, 1VIDA5, and LGP2; NOD-like receptors (NLRs),C-type lectin receptors (CLRs), and toll-like receptors (TLRs). In oneembodiment, the invention provides a nucleic acid molecule. Exemplarynucleic acids for use in this disclosure include ribonucleic acids(RNA), deoxyribonucleic acids (DNAs), peptide nucleic acids (PNAs),threose nucleic acids (TNAs), glycol nucleic acids (GNAs), lockednucleic acids (LNAs) or a hybrid thereof. As described herein, thenucleic acid molecule of the invention is not dependent on a particularnucleotide sequence. Rather, any nucleotide sequence may be used,provided that the sequence has the ability to form the structure of anucleic acid molecule described herein.

In one embodiment, the nucleic acid molecule of the invention comprisesa double stranded region. For example, in one embodiment, the nucleicacid molecule is a double stranded duplex. In one embodiment, thenucleic acid molecule of the invention is a single strand wherein afirst region of the molecule hybridizes with a second region of themolecule to form a duplex. In certain instances, the hairpin structureof the nucleic acid molecule may improve the stability of the duplex.

In one embodiment, the nucleic acid molecule comprises a blunt end. Inone embodiment, the nucleic acid molecule comprises a 5′ triphosphate ora 5′ diphosphate. In certain instances, the presence of one or more 5′triphosphate or 5′ diphosphate may improve the binding affinity of thenucleic acid molecule.

In one embodiment, the invention provides a nucleic acid molecule whichis capable of activating a PRR and inducing an IFN response in cellsexpressing a PRR. In one embodiment, the nucleic acid molecule of thepresent invention has a double-stranded section of less than 19 basepairs. In one embodiment, the nucleic acid molecule comprises at leastone 5′ triphosphate or at least one 5′ diphosphate. In one embodiment,the nucleic acid molecule comprises at least one blunt end.

The present invention encompasses the use of the nucleic acid moleculeto prevent and/or treat any disease, disorder, or condition in whichinducing IFN production would be beneficial. For example, increased IFNproduction, by way of the nucleic acid molecule of the invention, may bebeneficial to prevent or treat a wide variety of disorders, including,but not limited to, bacterial infection, viral infection, parasiticinfection, cancer, autoimmune diseases, respiratory disorders, and thelike.

In one embodiment, the invention provides a composition and method forthe prevention and/or treatment of a viral infection, including, but notlimited to, influenza, hepatitis, human papillomavirus, HIV, and thelike. In one embodiment, the invention provides a composition and methodfor the treatment of a cancer, including, but not limited to,hematological malignancies including various leukemias and lymphomas,carcinomas, blastomas, and sarcomas. In one embodiment, the inventionprovides a composition and method for the treatment of an autoimmunedisease, including but not limited to multiple sclerosis, psoriasis,arthritis, dermatitis, diabetes, lupus, colitis, Aicardi-Goutieressyndrome (AGS), and the like.

In one embodiment, the invention provides a composition and method forpreventing and/or treating a respiratory disorder, including, acute lunginjury (ALI), acute respiratory distress syndrome (ARDS), asthma,chronic obstructive pulmonary disease (COPD), obstructive sleep apnea(OSA), idiopathic pulmonary fibrosis (IPF), tuberculosis, pulmonaryhypertension, pleural effusion, and lung cancer.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “airway inflammation”, as used herein, means a disease orcondition related to inflammation on airway of subject. The airwayinflammation may be caused or accompanied by allergy(ies), asthma,impeded respiration, cystic fibrosis (CF), chronic obstructive pulmonarydiseases (COPD), allergic rhinitis (AR), acute respiratory distresssyndrome (ARDS), microbial or viral infections, pulmonary hypertension,lung inflammation, bronchitis, cancer, airway obstruction,bronchoconstriction, and the like.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addision's disease, alopecia greata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike.

The term “chronic obstructive pulmonary disease,” or COPD, is usedherein to refer to two lung diseases, chronic bronchitis and emphysema,that are characterized by obstruction to airflow that interferes withnormal breathing. Both of these conditions frequently co-exist.

“Complementary” refers to the broad concept of sequence complementaritybetween regions of two nucleic acid strands or between two regions ofthe same nucleic acid strand. It is known that an adenine residue of afirst nucleic acid region is capable of forming specific hydrogen bonds(“base pairing”) with a residue of a second nucleic acid region which isantiparallel to the first region if the residue is thymine or uracil.Similarly, it is known that a cytosine residue of a first nucleic acidstrand is capable of base pairing with a residue of a second nucleicacid strand which is antiparallel to the first strand if the residue isguanine. A first region of a nucleic acid is complementary to a secondregion of the same or a different nucleic acid if, when the two regionsare arranged in an antiparallel fashion, at least one nucleotide residueof the first region is capable of base pairing with a residue of thesecond region. Preferably, the first region comprises a first portionand the second region comprises a second portion, whereby, when thefirst and second portions are arranged in an antiparallel fashion, atleast about 50%, and preferably at least about 75%, at least about 90%,or at least about 95% of the nucleotide residues of the first portionare capable of base pairing with nucleotide residues in the secondportion. More preferably, all nucleotide residues of the first portionare capable of base pairing with nucleotide residues in the secondportion.

The term “emphysema” is a major subset of the clinical entity known asCOPD and is characterized by specific pathological changes in lungtissue over time. One hallmark of emphysema is the gradual, progressive,and irreversible destruction of the distal lung parenchyma leading tothe destruction alveoli. Alveolar destruction leads to enlargedairspaces in the lung and consequently a reduced ability to transferoxygen to the bloodstream. Emphysema is also characterized by a loss ofelasticity in the lung making it difficult to maintain open airways.Both of these changes produce the clinical sequelae of emphysemacomprising shortness of breath and difficulty exhaling, respectively.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA. Unless otherwise specified, a “nucleotide sequenceencoding an amino acid sequence” includes all nucleotide sequences thatare degenerate versions of each other and that encode the same aminoacid sequence. Nucleotide sequences that encode proteins and RNA mayinclude introns.

As used herein, the term “fragment,” as applied to a nucleic acid,refers to a subsequence of a larger nucleic acid. A “fragment” of anucleic acid can be at least about 5 nucleotides in length; for example,at least about 10 nucleotides to about 100 nucleotides; at least about100 to about 500 nucleotides, at least about 500 to about 1000nucleotides, at least about 1000 nucleotides to about 1500 nucleotides;or about 1500 nucleotides to about 2500 nucleotides; or about 2500nucleotides (and any integer value in between).

“Homologous, homology” or “identical, identity” as used herein, refer tocomparisons among amino acid and nucleic acid sequences. When referringto nucleic acid molecules, “homology,” “identity,” or “percentidentical” refers to the percent of the nucleotides of the subjectnucleic acid sequence that have been matched to identical nucleotides bya sequence analysis program. Homology can be readily calculated by knownmethods. Nucleic acid sequences and amino acid sequences can be comparedusing computer programs that align the similar sequences of the nucleicor amino acids and thus define the differences. In preferredmethodologies, the BLAST programs (NCBI) and parameters used therein areemployed, and the ExPaSy is used to align sequence fragments of genomicDNA sequences. However, equivalent alignment assessments can be obtainedthrough the use of any standard alignment software.

As used herein, “homologous” refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same subunit, e.g., if a position in eachof two DNA molecules is occupied by adenine, then they are homologous atthat position. The homology between two sequences is a direct functionof the number of matching or homologous positions, e.g., if half (e.g.,five positions in a polymer ten subunits in length) of the positions intwo compound sequences are homologous then the two sequences are 50%homologous, if 90% of the positions, e.g., 9 of 10, are matched orhomologous, the two sequences share 90% homology. By way of example, theDNA sequences 5′ATTGCC 3′ and 5′ TATGGC 3′ share 50% homology.

“Hybridization probes” are oligonucleotides capable of binding in abase-specific manner to a complementary strand of nucleic acid. Suchprobes include peptide nucleic acids, as described in Nielsen et al.,1991, Science 254, 1497-1500, and other nucleic acid analogs and nucleicacid mimetics. See U.S. Pat. No. 6,156,501.

The term “hybridization” refers to the process in which twosingle-stranded nucleic acids bind non-covalently to form adouble-stranded nucleic acid; triple-stranded hybridization is alsotheoretically possible. Complementary sequences in the nucleic acidspair with each other to form a double helix. The resultingdouble-stranded nucleic acid is a “hybrid.” Hybridization may bebetween, for example, two complementary or partially complementarysequences. The hybrid may have double-stranded regions and singlestranded regions. The hybrid may be, for example, DNA:DNA, RNA:DNA orDNA:RNA. Hybrids may also be formed between modified nucleic acids. Oneor both of the nucleic acids may be immobilized on a solid support.Hybridization techniques may be used to detect and isolate specificsequences, measure homology, or define other characteristics of one orboth strands.

The stability of a hybrid depends on a variety of factors including thelength of complementarity, the presence of mismatches within thecomplementary region, the temperature and the concentration of salt inthe reaction. Hybridizations are usually performed under stringentconditions, for example, at a salt concentration of no more than 1 M anda temperature of at least 25° C. For example, conditions of 5×SSPE (750mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) or 100 mM MES, 1 M Na,20 mM EDTA, 0.01% Tween-20 and a temperature of 25-50° C. are suitablefor allele-specific probe hybridizations. In a particularly preferredembodiment, hybridizations are performed at 40-50° C. Acetylated BSA andherring sperm DNA may be added to hybridization reactions. Hybridizationconditions suitable for microarrays are described in the Gene ExpressionTechnical Manual and the GeneChip Mapping Assay Manual available fromAffymetrix (Santa Clara, Calif.).

A first oligonucleotide anneals with a second oligonucleotide with “highstringency” if the two oligonucleotides anneal under conditions wherebyonly oligonucleotides which are at least about 75%, and preferably atleast about 90% or at least about 95%, complementary anneal with oneanother. The stringency of conditions used to anneal twooligonucleotides is a function of, among other factors, temperature,ionic strength of the annealing medium, the incubation period, thelength of the oligonucleotides, the G-C content of the oligonucleotides,and the expected degree of non-homology between the twooligonucleotides, if known. Methods of adjusting the stringency ofannealing conditions are known (see, e.g. Sambrook et al., 2012,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of a compound, composition, vector,or delivery system of the invention in the kit for effecting alleviationof the various diseases or disorders recited herein. Optionally, oralternately, the instructional material can describe one or more methodsof alleviating the diseases or disorders in a cell or a tissue of amammal. The instructional material of the kit of the invention can, forexample, be affixed to a container which contains the identifiedcompound, composition, vector, or delivery system of the invention or beshipped together with a container which contains the identifiedcompound, composition, vector, or delivery system. Alternatively, theinstructional material can be shipped separately from the container withthe intention that the instructional material and the compound be usedcooperatively by the recipient.

As used herein, “isolate” refers to a nucleic acid obtained from anindividual, or from a sample obtained from an individual. The nucleicacid may be analyzed at any time after it is obtained (e.g., before orafter laboratory culture, before or after amplification.)

The term “label” as used herein refers to a luminescent label, a lightscattering label or a radioactive label. Fluorescent labels include, butare not limited to, the commercially available fluoresceinphosphoramidites such as Fluoreprime (Pharmacia), Fluoredite (Millipore)and FAM (ABI). See U.S. Pat. No. 6,287,778.

The term “mismatch,” “mismatch control” or “mismatch probe” refers to anucleic acid whose sequence is not perfectly complementary to aparticular target sequence. The mismatch may comprise one or more bases.As used herein, the term “nucleic acid” refers to bothnaturally-occurring molecules such as DNA and RNA, but also variousderivatives and analogs. Generally, the probes, hairpin linkers, andtarget polynucleotides of the present teachings are nucleic acids, andtypically comprise DNA. Additional derivatives and analogs can beemployed as will be appreciated by one having ordinary skill in the art.

The term “nucleotide base,” as used herein, refers to a substituted orunsubstituted aromatic ring or rings. In certain embodiments, thearomatic ring or rings contain at least one nitrogen atom. In certainembodiments, the nucleotide base is capable of forming Watson-Crickand/or Hoogsteen hydrogen bonds with an appropriately complementarynucleotide base. Exemplary nucleotide bases and analogs thereof include,but are not limited to, naturally occurring nucleotide bases adenine,guanine, cytosine, 6 methyl-cytosine, uracil, thymine, and analogs ofthe naturally occurring nucleotide bases, e.g., 7-deazaadenine,7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, N6 delta2-isopentenyladenine (6iA), N6-delta 2-isopentenyl-2-methylthioadenine(2 ms6iA), N2-dimethylguanine (dmG), 7methylguanine (7 mG), inosine,nebularine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine,hypoxanthine, pseudouridine, pseudocytosine, pseudoisocytosine,5-propynylcytosine, isocytosine, isoguanine, 7-deazaguanine,2-thiopyrimidine, 6-thioguanine, 4-thiothymine, 4-thiouracil,06-methylguanine, N6-methyladenine, 04-methylthymine,5,6-dihydrothymine, 5,6-dihydrouracil, pyrazolo[3,4-D]pyrimidines (see,e.g., U.S. Pat. Nos. 6,143,877 and 6,127,121 and PCT publishedapplication WO 01/38584), ethenoadenine, indoles such as nitroindole and4-methylindole, and pyrroles such as nitropyrrole. Certain exemplarynucleotide bases can be found, e.g., in Fasman, 1989, Practical Handbookof Biochemistry and Molecular Biology, pp. 385-394, CRC Press, BocaRaton, Fla., and the references cited therein.

The term “nucleotide,” as used herein, refers to a compound comprising anucleotide base linked to the C-1′ carbon of a sugar, such as ribose,arabinose, xylose, and pyranose, and sugar analogs thereof. The termnucleotide also encompasses nucleotide analogs. The sugar may besubstituted or unsubstituted. Substituted ribose sugars include, but arenot limited to, those riboses in which one or more of the carbon atoms,for example the 2′-carbon atom, is substituted with one or more of thesame or different Cl, F, —R, —OR, —NR2 or halogen groups, where each Ris independently H, C1-C6 alkyl or C5-C14 aryl. Exemplary ribosesinclude, but are not limited to, 2′-(C1-C6)alkoxyribose,2′-(C5-C14)aryloxyribose, 2′,3′-didehydroribose, 2′-deoxy-3′-haloribose,2′-deoxy-3′-fluororibose, 2′-deoxy-3′-chlororibose,2′-deoxy-3′-aminoribose, 2′-deoxy-3′-(C1-C6)alkylribose,2′-deoxy-3′-(C1-C6)alkoxyribose and 2′-deoxy-3′-(C5-C14)aryloxyribose,ribose, 2′-deoxyribose, 2′,3′-dideoxyribose, 2′-haloribose,2′-fluororibose, 2′-chlororibose, and 2′-alkylribose, e.g., 2′-O-methyl,4′-anomeric nucleotides, 1′-anomeric nucleotides, 2′-4′- and3′-4′-linked and other “locked” or “LNA”, bicyclic sugar modifications(see, e.g., PCT published application nos. WO 98/22489, WO 98/39352; andWO 99/14226). The term “nucleic acid” typically refers to largepolynucleotides.

The term “oligonucleotide” typically refers to short polynucleotides,generally, no greater than about 50 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.”

The term “overhang,” as used herein, refers to terminal non-base pairingnucleotide(s) resulting from one strand or region extending beyond theterminus of the complementary strand to which the first strand or regionforms a duplex. One or more polynucleotides that are capable of forminga duplex through hydrogen bonding can have overhangs. Thesingle-stranded region extending beyond the 3′ end of the duplex isreferred to as an overhang.

The term “pattern recognition receptor,” abbreviated as PRR, as usedherein refers to a family of proteins that typically recognizepathogen-associated molecular patterns. PRRs may include members of theRIG-I like receptor (RLR) family, NOD-like receptor (NLRB) family,C-type lectin receptor (CLRs) family, or toll-like receptor (TLRs)family. In one embodiment of the present invention, the nucleic acidmolecule described herein binds to a PRR, thereby resulting in aninterferon response. It should be understood that a PRR includes any PRRfragment, variant, splice variant, mutant, or the like. In certainembodiments, the PRR is RIG-I.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning and amplification technology,and the like, and by synthetic means. An “oligonucleotide” as usedherein refers to a short polynucleotide, typically less than 100 basesin length.

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand”; sequences on the DNA strandwhich are located 5′ to a reference point on the DNA are referred to as“upstream sequences”; sequences on the DNA strand which are 3′ to areference point on the DNA are referred to as “downstream sequences.” Inthe sequences described herein: A=adenine, G=guanine, T=thymine,C=cytosine, U=uracil, H=A, C or T/U, R=A or G, M=A or C, K=G or T/U, S=Gor C, Y=C or T/U, W=A or T/U, B=G or C or T/U, D=A or G, or T/U, V=A orG or C, N=A or G or C or T/U.

The skilled artisan will understand that all nucleic acid sequences setforth herein throughout in their forward orientation, are also useful inthe compositions and methods of the invention in their reverseorientation, as well as in their forward and reverse complementaryorientation, and are described herein as well as if they were explicitlyset forth herein.

“Primer” refers to a polynucleotide that is capable of specificallyhybridizing to a designated polynucleotide template and providing apoint of initiation for synthesis of a complementary polynucleotide.Such synthesis occurs when the polynucleotide primer is placed underconditions in which synthesis is induced, e.g., in the presence ofnucleotides, a complementary polynucleotide template, and an agent forpolymerization such as DNA polymerase. A primer is typicallysingle-stranded, but may be double-stranded. Primers are typicallydeoxyribonucleic acids, but a wide variety of synthetic and naturallyoccurring primers are useful for many applications. A primer iscomplementary to the template to which it is designed to hybridize toserve as a site for the initiation of synthesis, but need not reflectthe exact sequence of the template. In such a case, specifichybridization of the primer to the template depends on the stringency ofthe hybridization conditions. Primers can be labeled with a detectablelabel, e.g., chromogenic, radioactive, or fluorescent moieties and usedas detectable moieties. Examples of fluorescent moieties include, butare not limited to, rare earth chelates (europium chelates), Texas Red,rhodamine, fluorescein, dansyl, phycocrytherin, phycocyanin, spectrumorange, spectrum green, and/or derivatives of any one or more of theabove. Other detectable moieties include digoxigenin and biotin.

As used herein a “probe” is defined as a nucleic acid capable of bindingto a target nucleic acid of complementary sequence through one or moretypes of chemical bonds, usually through complementary base pairing,usually through hydrogen bond formation. As used herein, a probe mayinclude natural (i.e. A, G, U, C, or T) or modified bases(7-deazaguanosine, inosine, etc.). In addition, a linkage other than aphosphodiester bond may join the bases in probes, so long as it does notinterfere with hybridization. Thus, probes may be peptide nucleic acidsin which the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. The term “match,” “perfect match,” “perfectmatch probe” or “perfect match control” refers to a nucleic acid thathas a sequence that is perfectly complementary to a particular targetsequence. The nucleic acid is typically perfectly complementary to aportion (subsequence) of the target sequence. A perfect match (PM) probecan be a “test probe”, a “normalization control” probe, an expressionlevel control probe and the like. A perfect match control or perfectmatch is, however, distinguished from a “mismatch” or “mismatch probe.”

The term “respiratory diseases”, as used herein, means diseases orconditions related to, the respiratory system. Examples include, but notlimited to, asthma, chronic obstructive pulmonary disease (COPD), airwayinflammation, allergy(ies), impeded respiration, cystic fibrosis (CF),allergic rhinitis (AR), acute respiratory distress syndrome (ARDS), lungcancer, pulmonary hypertension, lung inflammation, bronchitis, airwayobstruction, bronchoconstriction, microbial infection, and viralinfection, such as SARS. Other respiratory diseases referred to hereininclude dyspnea, emphysema, wheezing, pulmonary fibrosis,hyper-responsive airways, increased adenosine or adenosine receptorlevels, particularly those associated with infectious diseases,surfactant depletion, pulmonary vasoconstriction, impeded respiration,infantile respiratory distress syndrome (infantile RDS), allergicrhinitis, and the like.

The term “ribonucleotide” and the phrase “ribonucleic acid” (RNA), asused herein, refer to a modified or unmodified nucleotide orpolynucleotide comprising at least one ribonucleotide unit. Aribonucleotide unit comprises an oxygen attached to the 2′ position of aribosyl moiety having a nitrogenous base attached in N-glycosidiclinkage at the 1′ position of a ribosyl moiety, and a moiety that eitherallows for linkage to another nucleotide or precludes linkage.

The term “target” as used herein refers to a molecule that has anaffinity for a given molecule. Targets may be naturally-occurring orman-made molecules. Also, they can be employed in their unaltered stateor as aggregates with other species. Targets may be attached, covalentlyor noncovalently, to a binding member, either directly or via a specificbinding substance. Examples of targets which can be employed by thisinvention include, but are not restricted to, proteins, peptides,oligonucleotides and nucleic acids.

“Variant” as the term is used herein, is a nucleic acid sequence or apeptide sequence that differs in sequence from a reference nucleic acidsequence or peptide sequence respectively, but retains essentialproperties of the reference molecule. Changes in the sequence of anucleic acid variant may not alter the amino acid sequence of a peptideencoded by the reference nucleic acid, or may result in amino acidsubstitutions, additions, deletions, fusions and truncations. A variantof a nucleic acid or peptide can be a naturally occurring such as anallelic variant, or can be a variant that is not known to occurnaturally. Non-naturally occurring variants of nucleic acids andpeptides may be made by mutagenesis techniques or by direct synthesis.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

The present invention provides a nucleic acid molecule, for example ashort duplex nucleic acid molecule, which is capable of activating oneor more PRRs and inducing an IFN response in cells expressing a PRR. Inone embodiment, the nucleic acid molecule of the present inventioncomprises a double-stranded section of no more than 19 base pairs, andat least one blunt end. In one embodiment, the nucleic acid moleculecomprises a 5′ triphosphate or a 5′ diphosphate. In one embodiment, theinvention further provides the use of the nucleic acid molecule of theinvention for inducing an IFN response in vitro and in vivo. In oneembodiment, the nucleic acid molecule of the invention binds to RIG-I,or other PRRs, which in turn leads to increased IFN production.

Accordingly, the present invention provides the use of the nucleic acidmolecule of the invention for preventing and/or treating diseases orconditions in which inducing IFN production would be beneficial, such asinfections, tumors/cancers, inflammatory diseases, and disorders, andimmune disorders.

In one embodiment, the present invention provides the use of the nucleicacid molecule of the invention for assessing the level of expression,level of activity, or both of a PRR, or other members of the PRRpathway, in a cell. For example, in one embodiment, the inventionprovides a method of diagnosing a disease or disorder comprising usingthe nucleic acid molecule to assess the PRR-mediated IFN production in acell. In one embodiment, the invention provides a screening assay foridentifying a compound that alters PRR-mediated IFN response by usingthe nucleic acid molecule to assess PRR-mediated IFN response before,during, and/or after contact with a compound of interest.

In one embodiment, the nucleic acid of the invention comprisesintramolecular nucleotide base pairing (i.e., hairpin). Therefore, incertain aspects, the nucleic acid molecule of the invention is sometimesreferred herein as a short hairpin nucleic acid molecule.

The present invention encompasses compositions and method for inducingan interferon response produced by any PRR, including but not limited tomembers of the RIG-I like receptor (RLR) family; NOD-like receptor(NLRs) family, C-type lectin receptor (CLRs) family, and toll-likereceptor (TLRs) family. Thus, while in certain instances, the presentinvention is exemplified herein through stimulation of RIG-I, a skilledartisan would recognize that the present invention is equally applicableto the stimulation of any PRR known in the art, or discovered in thefuture.

Compositions

In one embodiment, the invention provides a nucleic acid molecule whichis capable of inducing an IFN response in cells expressing a PRR. In oneembodiment, the nucleic acid molecule of the present invention comprisesa double-stranded section of no more than 19 base pairs and at least oneblunt end. In one embodiment, the nucleic acid molecule comprises a 5′triphosphate or a 5′ diphosphate.

In one embodiment, the nucleic acid molecule of the present inventionhas a double-stranded section of less than 19 base pairs, in one aspectless than 18 base pairs, in one aspect less than 16 base pairs, in oneaspect less than 14 base pairs, in one aspect less than 12 base pairs,in one aspect less than 10 base pairs, in one aspect less than 8 basepairs, in one aspect less than 6 base pairs, in one aspect less than 4base pairs. In certain embodiments, the double-stranded sectioncomprises one or more mispaired bases. That is, Watson-Crick basepairing is not required at each and every nucleotide pair. In oneembodiment, the double-stranded section comprises about 4-19 base pairs.

In some instances, the nucleic acid molecule can be of any sequence andcomprises a hairpin structure and a blunt end, wherein the hairpincomprises a double-stranded section of less than 19 base pairs.

The nucleic acid molecule of the invention comprises nucleic acids fromany source. A nucleic acid in the context of the present inventionincludes but is not limited to deoxyribonucleic acid (DNA), ribonucleicacid (RNA), peptide nucleic acid (PNA, threose nucleic acid (TNA),glycol nucleic acid (GNA), locked nucleic acid (LNA) or a hybridthereof.). DNA and RNA are naturally occurring in organisms, however,they may also exist outside living organisms or may be added toorganisms. The nucleic acid may be of any origin, e.g., viral,bacterial, archae-bacterial, fungal, ribosomal, eukaryotic orprokaryotic. It may be nucleic acid from any biological sample and anyorganism, tissue, cell or sub-cellular compartment. It may be nucleicacid from any organism. The nucleic acid may be pre-treated beforequantification, e.g., by isolation, purification or modification. Alsoartificial or synthetic nucleic acid may be used. The length of thenucleic acids may vary. The nucleic acids may be modified, e.g. maycomprise one or more modified nucleobases or modified sugar moieties(e.g., comprising methoxy groups). The backbone of the nucleic acid maycomprise one or more peptide bonds as in peptide nucleic acid (PNA). Thenucleic acid may comprise a base analog such as non-purine ornon-pyrimidine analog or nucleotide analog. It may also compriseadditional attachments such as proteins, peptides and/or or amino acids.

In one embodiment, the nucleic acid molecule of the invention is asingle stranded oligonucleotide that forms an intramolecular structure,i.e., a hairpin structure.

In one embodiment, the hairpin nucleic acid molecule forms a blunt end.In one embodiment, a blunt end refers to refers to, e.g., an RNA duplexwhere at least one end of the duplex lacks any overhang, e.g., a 3′dinucleotide overhang, such that both the 5′ and 3′ strand end together,i.e., are flush or as referred to herein, are blunt. The molecules ofthe invention have at least one blunt end. In some instances, theintramolecular structure produces a 3′ overhang. In some instances, theintramolecular structure produces a 5′ overhang.

In certain instances, the short hairpin nucleic acid molecule of theinvention is an ideal stimulant because of the ability to re-annealafter being unwound, whereas the shorter palindromic duplexes that arenot a hairpin would likely lose their ability to stimulate IFNproduction as soon as the duplex melted. However, the present inventionis not limited to hairpin structures, as it is demonstrated herein thatshort double-stranded duplexes demonstrate the ability to bind to a PRRand stimulate an interferon response.

In some instances, the short hairpin nucleic acid molecule of theinvention is designed that in some conditions, the intramolecular stemstructure has reduced stability where the stem structure is unfolded. Inthis manner, the stem structure can be designed so that the stemstructure can be relieved of its intramolecular base pairing andresemble more of a linear molecule.

In accordance with the present invention, there are providedpredetermined stem oligonucleotide sequences containing stretches ofcomplementary sequences that form the stem structure. In one embodiment,the stem comprises a double-stranded section that comprise in one aspectless than 19 base pairs, in one aspect less than 18 base pairs, in oneaspect less than 16 base pairs, in one aspect less than 14 base pairs,in one aspect less than 12 base pairs, in one aspect less than 10 basepairs, in one aspect less than 8 base pairs, in one aspect less than 6base pairs, in one aspect less than 4 base pairs, such that thesecomplementary stretches anneal to provide a hairpin structure. In oneembodiment, the double-stranded section comprises one or more basemispairs. That is, the double-stranded section need not compriseWatson-Crick base pairing at each and every base pair in order toproduce the hairpin structure.

In one embodiment, the short hairpin nucleic acid molecule of theinvention comprising: an antisense sequence and a sense sequence,wherein the sense sequence is substantially complementary to theantisense sequence; and a loop region connecting the antisense and sensesequences.

In certain aspects, the present invention includes a polynucleotidecomprising a unimolecular RNA, such as a short hairpin RNA. The shorthairpin RNA can be a unimolecular RNA that includes a sense sequence, aloop region, and an antisense sequence which together form a hairpinloop structure. Preferably, the antisense and sense sequences aresubstantially complementary to one other (about 80% complementary ormore), where in certain embodiments the antisense and sense sequencesare 100% complementary to each other. In certain embodiments, antisenseand sense sequences each comprises less than 19 nucleotides in length,e.g., between 18 and 8 nucleotides in length. Additionally, theantisense and sense sequences within a unimolecular RNA of the inventioncan be the same length or differ in length. The loop can be any length,for example a length being 0, 1 or more, 2 or more, 4 or more, 5 ormore, 8 or more, 10 or more, 15 or more, 20 or more, 40 or more, or 100or more nucleotides in length.

Nucleic Acid Modification

The nucleic acid molecules of the present invention can be modified toimprove stability in serum or in growth medium for cell cultures. Inorder to enhance the stability, the 3′-residues may be stabilizedagainst degradation, e.g., they may be selected such that they consistof purine nucleotides, particularly adenosine or guanosine nucleotides.Alternatively, substitution of pyrimidine nucleotides by modifiedanalogues, e.g., substitution of uridine by 2′-deoxythymidine istolerated and does not affect function of the molecule.

In one embodiment of the present invention the nucleic acid molecule maycontain at least one modified nucleotide analogue. For example, the endsmay be stabilized by incorporating modified nucleotide analogues.

Non-limiting examples of nucleotide analogues include sugar- and/orbackbone-modified ribonucleotides (i.e., include modifications to thephosphate-sugar backbone). For example, the phosphodiester linkages ofnatural RNA may be modified to include at least one of a nitrogen orsulfur heteroatom. In preferred backbone-modified ribonucleotides thephosphoester group connecting to adjacent ribonucleotides is replaced bya modified group, e.g., of phosphothioate group. In preferredsugar-modified ribonucleotides, the 2′ OH-group is replaced by a groupselected from H, OR, R, halo, SH, SR, NH₂, NHR, NR₂ or ON, wherein R isC₁-C₆ alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

Other examples of modifications are nucleobase-modified ribonucleotides,i.e., ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

Modifications can be added to enhance stability, functionality, and/orspecificity and to minimize immunostimulatory properties of the shorthairpin nucleic acid molecule of the invention. For example, theoverhangs can be unmodified, or can contain one or more specificity orstabilizing modifications, such as a halogen or O-alkyl modification ofthe 2′ position, or internucleotide modifications such asphosphorothioate modification. The overhangs can be ribonucleic acid,deoxyribonucleic acid, or a combination of ribonucleic acid anddeoxyribonucleic acid.

In some instances, the nucleic acid molecule comprises at least one ofthe following chemical modifications: 2′-H, 2′-O-methyl, or 2′-OHmodification of one or more nucleotides; one or more phosphorothioatemodifications of the backbone; and a non-nucleotide moiety; wherein theat least one chemical modification confers reduced immunostimulatoryactivity, increased serum stability, or both, as compared to acorresponding short hairpin nucleic acid molecule not having thechemical modification.

In certain embodiments, the pyrimidine nucleotides comprise2′-O-methylpyrimidine nucleotides and/or 2′-deoxy-pyrimidinenucleotides.

In certain embodiments, some or all of the purine nucleotides cancomprise 2′-O-methylpurine nucleotides and/or 2′-deoxy-purinenucleotides.

In certain embodiments, the chemical modification is present innucleotides proximal to the 3′ and/or 5′ ends of the nucleic acidmolecule of the invention.

In certain embodiments, a nucleic acid molecule of the invention canhave enhanced resistance to nucleases. For increased nucleaseresistance, a nucleic acid molecule, can include, for example,2′-modified ribose units and/or phosphorothioate linkages. For example,the 2′ hydroxyl group (OH) can be modified or replaced with a number ofdifferent “oxy” or “deoxy” substituents.

For increased nuclease resistance the nucleic acid molecules of theinvention can include 2′-O-methyl, 2′-fluorine, 2′-O-methoxyethyl,2′-O-aminopropyl, 2′-amino, and/or phosphorothioate linkages. Inclusionof locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g.,2′-4′-ethylene-bridged nucleic acids, and certain nucleobasemodifications such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clampmodifications, can also increase binding affinity to a target.

In one embodiment, the nucleic acid molecule includes a 2′-modifiednucleotide, e.g., a 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA). In one embodiment, the nucleic acidmolecule includes at least one 2′-O-methyl-modified nucleotide, and insome embodiments, all of the nucleotides of the nucleic acid moleculeinclude a 2′-O-methyl modification.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; amine,0-AMINE and aminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroarylamino, or diheteroaryl amino, ethylene diamine, polyamino). It isnoteworthy that oligonucleotides containing only the methoxyethyl group(MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nuclease stabilitiescomparable to those modified with the robust phosphorothioatemodification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars); halo(e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino, heterocyclyl,arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or aminoacid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroarylamino, or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl, aryl,aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino functionality.

Preferred substitutents are 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl,2′-C-allyl, and 2′-fluoro.

One way to increase resistance is to identify cleavage sites and modifysuch sites to inhibit cleavage. For example, the dinucleotides 5′-UA-3′,5′-UG-3′, 5′-CA-3′, 5′-UU-3′, or 5′-CC-3′ can serve as cleavage sites.Enhanced nuclease resistance can therefore be achieved by modifying the5′ nucleotide, resulting, for example, in at least one5′-uridine-adenine-3′ (5′-UA-3′) dinucleotide wherein the uridine is a2′-modified nucleotide; at least one 5′-uridine-guanine-3′ (5′-UG-3′)dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide; atleast one 5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, wherein the5′-cytidine is a 2′-modified nucleotide; at least one5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide; or at least one 5′-cytidine-cytidine-3′(5′-CC-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modifiednucleotide. The oligonucleotide molecule can include at least 2, atleast 3, at least 4 or at least 5 of such dinucleotides. In certainembodiments, all the pyrimidines of a nucleic acid molecule carry a2′-modification, and the nucleic acid molecule therefore has enhancedresistance to endonucleases.

With respect to phosphorothioate linkages that serve to increaseprotection against RNase activity, the nucleic acid molecule can includea phosphorothioate at least the first, second, or third internucleotidelinkage at the 5′ or 3′ end of the nucleotide sequence. To maximizenuclease resistance, the 2′ modifications can be used in combinationwith one or more phosphate linker modifications (e.g.,phosphorothioate).

In certain embodiments, the inclusion of pyranose sugars in the nucleicacid backbone can also decrease endonucleolytic cleavage. The certainembodiments, inclusion of furanose sugars in the nucleic acid backbonecan also decrease endonucleolytic cleavage.

In certain embodiments, the 5′-terminus can be blocked with anaminoalkyl group, e.g., a 5′-O-alkylamino substituent. Other 5′conjugates can inhibit 5′-3′ exonucleolytic cleavage. While not beingbound by theory, a 5′ conjugate, may inhibit exonucleolytic cleavage bysterically blocking the exonuclease from binding to the 5′-end ofoligonucleotide. Even small alkyl chains, aryl groups, or heterocyclicconjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) canblock 5′-3--exonucleases.

Thus, a nucleic acid molecule can include modifications so as to inhibitdegradation, e.g., by nucleases, e.g., endonucleases or exonucleases,found in the body of a subject. These monomers are referred to herein asNRMs, or Nuclease Resistance promoting Monomers, the correspondingmodifications as NRM modifications. In many cases these modificationswill modulate other properties of the oligonucleotide molecule as well,e.g., the ability to interact with a protein, e.g., a transport protein,e.g., serum albumin.

One or more different NRM modifications can be introduced into a nucleicacid molecule or into a sequence of a nucleic acid molecule. An NRMmodification can be used more than once in a sequence or in a nucleicacid molecule.

NRM modifications include some which can be placed only at the terminusand others which can go at any position. Some NRM modifications that caninhibit hybridization are preferably used only in terminal regions, andmore preferably not at the cleavage site or in the cleavage region of anucleic acid molecule.

Such modifications can be introduced into the terminal regions, e.g., atthe terminal position or with 2, 3, 4, or 5 positions of the terminus,of a sequence which targets or a sequence which does not target asequence in the subject.

In one embodiment, a nucleic acid molecule, includes a modification thatimproves targeting, e.g. a targeting modification described herein.Examples of modifications that target a nucleic acid molecule toparticular cell types include carbohydrate sugars such as galactose,N-acetylgalactosamine, mannose; vitamins such as folates; other ligandssuch as RGDs and RGD mimics; and small molecules including naproxen,ibuprofen or other known protein-binding molecules.

A nucleic acid molecule can be constructed using chemical synthesisand/or enzymatic ligation reactions using procedures known in the art.For example, a nucleic acid molecule can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the binding between the nucleic acidmolecule and target, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Other appropriate nucleic acidmodifications are described herein. Alternatively, the nucleic acidmolecule can be produced biologically using an expression vector.

The term “halo” refers to any radical of fluorine, chlorine, bromine oriodine.

The term “alkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms. For example, C₁-C₁₂ alkyl indicates that the group may have from1 to 12 (inclusive) carbon atoms in it. The term “haloalkyl” refers toan alkyl in which one or more hydrogen atoms are replaced by halo, andincludes alkyl moieties in which all hydrogens have been replaced byhalo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may beoptionally inserted with O, N, or S. The terms “aralkyl” refers to analkyl moiety in which an alkyl hydrogen atom is replaced by an arylgroup. Aralkyl includes groups in which more than one hydrogen atom hasbeen replaced by an aryl group. Examples of “aralkyl” include benzyl,9-fluorenyl, benzhydryl, and trityl groups.

The term “alkenyl” refers to a straight or branched hydrocarbon chaincontaining 2-8 carbon atoms and characterized in having one or moredouble bonds. Examples of a typical alkenyl include, but not limited to,allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. The term“alkynyl” refers to a straight or branched hydrocarbon chain containing2-8 carbon atoms and characterized in having one or more triple bonds.Some examples of a typical alkynyl are ethynyl, 2-propynyl, and3-methylbutynyl, and propargyl. The sp² and sp³ carbons may optionallyserve as the point of attachment of the alkenyl and alkynyl groups,respectively.

The terms “alkylamino” and “dialkylamino” refer to —NH(alkyl) and—NH(alkyl)₂ radicals respectively. The term “aralkylamino” refers to a—NH(aralkyl) radical. The term “alkoxy” refers to an —O-alkyl radical,and the terms “cycloalkoxy” and “aralkoxy” refer to an —O-cycloalkyl andO-aralkyl radicals respectively. The term “siloxy” refers to a R₃SiO—radical. The term “mercapto” refers to an SH radical. The term“thioalkoxy” refers to an —S-alkyl radical.

The term “alkylene” refers to a divalent alkyl (i.e., —R—), e.g., —CH₂—,—CH₂CH₂—, and —CH₂CH₂CH₂—. The term “alkylenedioxo” refers to a divalentspecies of the structure —O—R—O—, in which R represents an alkylene.

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system, wherein any ring atom can be substituted.Examples of aryl moieties include, but are not limited to, phenyl,naphthyl, anthracenyl, and pyrenyl.

The term “cycloalkyl” as employed herein includes saturated cyclic,bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 12carbons, wherein any ring atom can be substituted. The cycloalkyl groupsherein described may also contain fused rings. Fused rings are ringsthat share a common carbon-carbon bond or a common carbon atom (e.g.,spiro-fused rings). Examples of cycloalkyl moieties include, but are notlimited to, cyclohexyl, adamantyl, and norbornyl, and decalin.

The term “heterocyclyl” refers to a nonaromatic 3-10 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms ofN, O, or S if monocyclic, bicyclic, or tricyclic, respectively), whereinany ring atom can be substituted. The heterocyclyl groups hereindescribed may also contain fused rings. Fused rings are rings that sharea common carbon-carbon bond or a common carbon atom (e.g., spiro-fusedrings). Examples of heterocyclyl include, but are not limited totetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino,pyrrolinyl and pyrrolidinyl.

The term “cycloalkenyl” as employed herein includes partiallyunsaturated, nonaromatic, cyclic, bicyclic, tricyclic, or polycyclichydrocarbon groups having 5 to 12 carbons, preferably 5 to 8 carbons,wherein any ring atom can be substituted. The cycloalkenyl groups hereindescribed may also contain fused rings. Fused rings are rings that sharea common carbon-carbon bond or a common carbon atom (e.g., spiro-fusedrings). Examples of cycloalkenyl moieties include, but are not limitedto cyclohexenyl, cyclohexadienyl, or norbornenyl.

The term “heterocycloalkenyl” refers to a partially saturated,nonaromatic 5-10 membered monocyclic, 8-12 membered bicyclic, or 11-14membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, saidheteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6,or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,respectively), wherein any ring atom can be substituted. Theheterocycloalkenyl groups herein described may also contain fused rings.Fused rings are rings that share a common carbon-carbon bond or a commoncarbon atom (e.g., spiro-fused rings). Examples of heterocycloalkenylinclude but are not limited to tetrahydropyridyl and dihydropyran.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein any ring atomcan be substituted. The heteroaryl groups herein described may alsocontain fused rings that share a common carbon-carbon bond.

The term “oxo” refers to an oxygen atom, which forms a carbonyl whenattached to carbon, an N-oxide when attached to nitrogen, and asulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further substituted by substituents.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl,cycloalkenyl, aryl, or heteroaryl group at any atom of that group.Suitable substituents include, without limitation, alkyl, alkenyl,alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO₃H, sulfate,phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy,ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl),S(O)_(n)alkyl (where n is 0-2), S(O)_(n) aryl (where n is 0-2), S(O)_(n)heteroaryl (where n is 0-2), S(O)_(n) heterocyclyl (where n is 0-2),amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, andcombinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide(mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof),sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinationsthereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstitutedheterocyclyl, and unsubstituted cycloalkyl. In one aspect, thesubstituents on a group are independently any one single, or any subsetof the aforementioned substituents. The terms “adeninyl, cytosinyl,guaninyl, thyminyl, and uracilyl” and the like refer to radicals ofadenine, cytosine, guanine, thymine, and uracil.

A “protected” moiety refers to a reactive functional group, e.g., ahydroxyl group or an amino group, or a class of molecules, e.g., sugars,having one or more functional groups, in which the reactivity of thefunctional group is temporarily blocked by the presence of an attachedprotecting group. Protecting groups useful for the monomers and methodsdescribed herein can be found, e.g., in Greene, T. W., Protective Groupsin Organic Synthesis (John Wiley and Sons: New York), 1981, which ishereby incorporated by reference.

For ease of exposition the term nucleotide or ribonucleotide issometimes used herein in reference to one or more monomeric subunits ofan oligonucleotide agent. It will be understood herein that the usage ofthe term “ribonucleotide” or “nucleotide” herein can, in the case of amodified RNA or nucleotide surrogate, also refer to a modifiednucleotide, or surrogate replacement moiety at one or more positions.

In certain embodiments, the nucleic acid molecule of the inventionpreferably has one or more of the following properties:

(1) a 5′ modification that includes one or more phosphate groups or oneor more analogs of a phosphate group;(2) despite modifications, even to a very large number of basesspecifically base pair and form a duplex structure with adouble-stranded region;(3) despite modifications, even to a very large number, or all of thenucleosides, still have “RNA-like” properties, i.e., it will possess theoverall structural, chemical and physical properties of an RNA molecule,even though not exclusively, or even partly, of ribonucleotide-basedcontent. For example, all of the nucleotide sugars can contain e.g.,2′OMe, 2′fluoro in place of 2′ hydroxyl. Thisdeoxyribonucleotide-containing agent can still be expected to exhibitRNA-like properties. While not wishing to be bound by theory, anelectronegative fluorine prefers an axial orientation when attached tothe C2′ position of ribose. This spatial preference of fluorine can, inturn, force the sugars to adopt a C3′-endo pucker. This is the samepuckering mode as observed in RNA molecules and gives rise to theRNA-characteristic A-family-type helix. Further, since fluorine is agood hydrogen bond acceptor, it can participate in the same hydrogenbonding interactions with water molecules that are known to stabilizeRNA structures. (Generally, it is preferred that a modified moiety atthe 2′ sugar position will be able to enter into hydrogen-bonding whichis more characteristic of the 2′-OH moiety of a ribonucleotide than the2′-H moiety of a deoxyribonucleotide. In one embodiment, theoligonucleotide molecule will: exhibit a C3′-endo pucker in all, or atleast 50, 75, 80, 85, 90, or 95% of its sugars; exhibit a C3′-endopucker in a sufficient amount of its sugars that it can give rise to athe RNA-characteristic A-family-type helix; will have no more than 20,10, 5, 4, 3, 2, or 1 sugar which is not a C3′-endo pucker structure.

2′-modifications with C3′-endo sugar pucker include 2′-OH, 2′-O-Me,2′-O-methoxyethyl, 2′-O-aminopropyl, 2′-F, 2′-O-CH2-CO—NHMe,2′-O-CH2-CH2-O-CH2-CH2-N(Me)2, and LNA.

2′-modifications with a C2′-endo sugar pucker include 2′-H, 2′-Me,2′-S-Me, 2′-Ethynyl, 2′-ara-F.

Sugar modifications can also include L-sugars and 2′-5′-linked sugars.

Nucleic acid agents discussed herein include otherwise unmodified RNAand DNA as well as RNA and DNA that have been modified, e.g., to improveefficacy, and polymers of nucleoside surrogates. Unmodified RNA refersto a molecule in which the components of the nucleic acid, namelysugars, bases, and phosphate moieties, are the same or essentially thesame as that which occur in nature, preferably as occur naturally in thehuman body. The art has referred to rare or unusual, but naturallyoccurring, RNAs as modified RNAs, see, e.g., Limbach et al. (NucleicAcids Res., 1994, 22:2183-2196). Such rare or unusual RNAs, often termedmodified RNAs, are typically the result of a post-transcriptionalmodification and are within the term unmodified RNA as used herein.Modified RNA, as used herein, refers to a molecule in which one or moreof the components of the nucleic acid, namely sugars, bases, andphosphate moieties, are different from that which occur in nature,preferably different from that which occurs in the human body. Whilethey are referred to as “modified RNAs” they will of course, because ofthe modification, include molecules that are not, strictly speaking,RNAs. Nucleoside surrogates are molecules in which the ribophosphatebackbone is replaced with a non-ribophosphate construct that allows thebases to be presented in the correct spatial relationship such thathybridization is substantially similar to what is seen with aribophosphate backbone, e.g., non-charged mimics of the ribophosphatebackbone. Examples of all of the above are discussed herein.

As nucleic acids are polymers of subunits or monomers, many of themodifications described below occur at a position which is repeatedwithin a nucleic acid, e.g., a modification of a base, or a phosphatemoiety, or a non-linking 0 of a phosphate moiety. In some cases themodification will occur at all of the subject positions in the nucleicacid but in many, and in fact in most cases it will not. By way ofexample, a modification may only occur at a 3′ or 5′ terminal position,in a terminal region, e.g., at a position on a terminal nucleotide, orin the last 2, 3, 4, 5, or 10 nucleotides of a strand. The ligand can beattached at the 3′ end, the 5′ end, or at an internal position, or at acombination of these positions. For example, the ligand can be at the 3′end and the 5′ end; at the 3′ end and at one or more internal positions;at the 5′ end and at one or more internal positions; or at the 3′ end,the 5′ end, and at one or more internal positions. For example, aphosphorothioate modification at a non-linking 0 position may only occurat one or both termini, or may only occur in a terminal region, e.g., ata position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10nucleotides of the nucleic acid. The 5′ end can be phosphorylated.

Modifications and nucleotide surrogates are discussed below.

The scaffold presented above in Formula 1 represents a portion of aribonucleic acid. The basic components are the ribose sugar, the base,the terminal phosphates, and phosphate internucleotide linkers. Wherethe bases are naturally occurring bases, e.g., adenine, uracil, guanineor cytosine, the sugars are the unmodified 2′ hydroxyl ribose sugar (asdepicted) and W, X, Y, and Z are all O, Formula 1 represents a naturallyoccurring unmodified oligoribonucleotide.

Unmodified oligoribonucleotides may be less than optimal in someapplications, e.g., unmodified oligoribonucleotides can be prone todegradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleicacid phosphodiester bonds. However, chemical modifications to one ormore of the above RNA components can confer improved properties, and,for example, can render oligoribonucleotides more stable to nucleases.Unmodified oligoribonucleotides may also be less than optimal in termsof offering tethering points for attaching ligands or other moieties toa nucleic acid agent.

Modified nucleic acids and nucleotide surrogates can include one or moreof:

(i) alteration, e.g., replacement, of one or both of the non-linking (Xand Y) phosphate oxygens and/or of one or more of the linking (W and Z)phosphate oxygens (When the phosphate is in the terminal position, oneof the positions W or Z will not link the phosphate to an additionalelement in a naturally occurring ribonucleic acid. However, forsimplicity of terminology, except where otherwise noted, the W positionat the 5′ end of a nucleic acid and the terminal Z position at the 3′end of a nucleic acid, are within the term “linking phosphate oxygens”as used herein.);(ii) alteration, e.g., replacement, of a constituent of the ribosesugar, e.g., of the 2′ hydroxyl on the ribose sugar, or wholesalereplacement of the ribose sugar with a structure other than ribose,e.g., as described herein;(iii) wholesale replacement of the phosphate moiety (bracket I) with“dephospho” linkers;(iv) modification or replacement of a naturally occurring base;(v) replacement or modification of the ribose-phosphate backbone(bracket II);(vi) modification of the 3′ end or 5′ end of the RNA, e.g., removal,modification or replacement of a terminal phosphate group or conjugationof a moiety, such as a fluorescently labeled moiety, to either the 3′ or5′ end of RNA.

The terms replacement, modification, alteration, and the like, as usedin this context, do not imply any process limitation, e.g., modificationdoes not mean that one must start with a reference or naturallyoccurring ribonucleic acid and modify it to produce a modifiedribonucleic acid but rather modified simply indicates a difference froma naturally occurring molecule. It is understood that the actualelectronic structure of some chemical entities cannot be adequatelyrepresented by only one canonical form (i.e. Lewis structure). While notwishing to be bound by theory, the actual structure can instead be somehybrid or weighted average of two or more canonical forms, knowncollectively as resonance forms or structures. Resonance structures arenot discrete chemical entities and exist only on paper. They differ fromone another only in the placement or “localization” of the bonding andnonbonding electrons for a particular chemical entity. It can bepossible for one resonance structure to contribute to a greater extentto the hybrid than the others. Thus, the written and graphicaldescriptions of the embodiments of the present invention are made interms of what the art recognizes as the predominant resonance form for aparticular species. For example, any phosphoroamidate (replacement of anonlinking oxygen with nitrogen) would be represented by X═O and Y═N inthe above figure.

Specific modifications are discussed in more detail below.

The Phosphate Group

The phosphate group is a negatively charged species. The charge isdistributed equally over the two non-linking oxygen atoms (i.e., X and Yin Formula 1 above). However, the phosphate group can be modified byreplacing at least one of the oxygens with a different substituent. Oneresult of this modification to RNA phosphate backbones can be increasedresistance of the oligoribonucleotide to nucleolytic breakdown. Thuswhile not wishing to be bound by theory, it can be desirable in someembodiments to introduce alterations which result in either an unchargedlinker or a charged linker with unsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulfur. Unlike the situation where only one of X or Y isaltered, the phosphorus center in the phosphorodithioates is achiralwhich precludes the formation of oligoribonucleotides diastereomers.Diastereomer formation can result in a preparation in which theindividual diastereomers exhibit varying resistance to nucleases.Further, the hybridization affinity of RNA containing chiral phosphategroups can be lower relative to the corresponding unmodified RNAspecies. Thus, while not wishing to be bound by theory, modifications toboth X and Y which eliminate the chiral center, e.g., phosphorodithioateformation, may be desirable in that they cannot produce diastereomermixtures. Thus, X can be any one of S, Se, B, C, H, N, or OR (R is alkylor aryl). Thus Y can be any one of S, Se, B, C, H, N, or OR (R is alkylor aryl). Replacement of X and/or Y with sulfur is preferred.

The phosphate linker can also be modified by replacement of a linkingoxygen (i.e., W or Z in Formula 1) with nitrogen (bridgedphosphoroamidates), sulfur (bridged phosphorothioates) and carbon(bridged methylenephosphonates). The replacement can occur at a terminaloxygen (position W (3′) or position Z (5′)). Replacement of W withcarbon or Z with nitrogen is preferred.

Candidate agents can be evaluated for suitability as described below.

The Sugar Group

A modified RNA can include modification of all or some of the sugargroups of the ribonucleic acid. For example, the 2′ hydroxyl group (OH)can be modified or replaced with a number of different “oxy” or “deoxy”substituents. While not being bound by theory, enhanced stability isexpected since the hydroxyl can no longer be deprotonated to form a 2′alkoxide ion. The 2′ alkoxide can catalyze degradation by intramolecularnucleophilic attack on the linker phosphorus atom. Again, while notwishing to be bound by theory, it can be desirable to some embodimentsto introduce alterations in which alkoxide formation at the 2′ positionis not possible.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge or ethylene bridge (e.g., 2′-4′-ethylene bridgednucleic acid (ENA)), to the 4′ carbon of the same ribose sugar; amino,O-AMINE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino,diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine,polyamino) and aminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino).It is noteworthy that oligonucleotides containing only the methoxyethylgroup (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nucleasestabilities comparable to those modified with the robustphosphorothioate modification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars); halo(e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino, heterocyclyl,arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or aminoacid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl,heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; andalkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionallysubstituted with e.g., an amino functionality. Preferred substitutentsare 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl, 2′-C-allyl, and 2′-fluoro.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified RNA can include nucleotidescontaining e.g., arabinose, as the sugar. Modified RNAs can also include“abasic” sugars, which lack a nucleobase at C-1′. These abasic sugarscan also contain modifications at one or more of the constituent sugaratoms.

To maximize nuclease resistance, the 2′ modifications can be used incombination with one or more phosphate linker modifications (e.g.,phosphorothioate). The so-called “chimeric” oligonucleotides are thosethat contain two or more different modifications.

The modification can also entail the wholesale replacement of a ribosestructure with another entity (an SRMS) at one or more sites in thenucleic acid agent.

Candidate modifications can be evaluated as described below.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containingconnectors (cf. Bracket I in Formula 1 above). While not wishing to bebound by theory, it is believed that since the charged phosphodiestergroup is the reaction center in nucleolytic degradation, its replacementwith neutral structural mimics should impart enhanced nucleasestability. Again, while not wishing to be bound by theory, it can bedesirable, in some embodiment, to introduce alterations in which thecharged phosphate group is replaced by a neutral moiety.

Examples of moieties which can replace the phosphate group includesiloxane, carbonate, carboxymethyl, carbamate, amide, thioether,ethylene oxide linker, sulfonate, sulfonamide, thioformacetal,formacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.Preferred replacements include the methylenecarbonylamino andmethylenemethylimino groups.

Candidate modifications can be evaluated as described below.

Replacement of Ribophosphate Backbone

Oligonucleotide-mimicking scaffolds can also be constructed wherein thephosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates (see Bracket II of Formula 1 above).While not wishing to be bound by theory, it is believed that the absenceof a repetitively charged backbone diminishes binding to proteins thatrecognize polyanions (e.g. nucleases). Again, while not wishing to bebound by theory, it can be desirable in some embodiment, to introducealterations in which the bases are tethered by a neutral surrogatebackbone.

Examples include the mophilino, cyclobutyl, pyrrolidine and peptidenucleic acid (PNA) nucleoside surrogates. A preferred surrogate is a PNAsurrogate. Candidate modifications can be evaluated as described below.

Terminal Modifications

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end, 5′ end or both ends of the molecule.They can include modification or replacement of an entire terminalphosphate or of one or more of the atoms of the phosphate group. E.g.,the 3′ and 5′ ends of an oligonucleotide can be conjugated to otherfunctional molecular entities such as labeling moieties, e.g.,fluorophores (e.g., pyrene, TAIVIRA, fluorescein, Cy3 or Cy5 dyes) orprotecting groups (based e.g., on sulfur, silicon, boron or ester). Thefunctional molecular entities can be attached to the sugar through aphosphate group and/or a spacer. The terminal atom of the spacer canconnect to or replace the linking atom of the phosphate group or theC-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, the spacercan connect to or replace the terminal atom of a nucleotide surrogate(e.g., PNAs). These spacers or linkers can include e.g., —(CH₂)_(n)—,—(CH₂)_(n)N—, —(CH₂)_(n)O—, —(CH₂)_(n)S—, O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g.,n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine,thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotinand fluorescein reagents. While not wishing to be bound by theory, it isbelieved that conjugation of certain moieties can improve transport,hybridization, and specificity properties. Again, while not wishing tobe bound by theory, it may be desirable to introduce terminalalterations that improve nuclease resistance. Other examples of terminalmodifications include dyes, intercalating agents (e.g. acridines),cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic carriers (e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles).

Terminal modifications can be added for a number of reasons, includingas discussed elsewhere herein to modulate activity or to modulateresistance to degradation. Preferred modifications include the additionof a methylphosphonate at the 3′-most terminal linkage; a 3′C5-aminoalkyl-dT; 3′ cationic group; or another 3′ conjugate to inhibit3′-5′ exonucleolytic degradation.

Terminal modifications useful for modulating activity includemodification of the 5′ end with phosphate or phosphate analogs. Forexample, in certain embodiments, oligonucleotide agents are 5′phosphorylated or include a phosphoryl analog at the 5′ terminus.Suitable modifications include: 5′-monophosphate ((HO)2(O)P—O-5′);5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-).

Terminal modifications can also be useful for monitoring distribution,and in such cases the preferred groups to be added include fluorophores,e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminalmodifications can also be useful for enhancing uptake, usefulmodifications for this include cholesterol. Terminal modifications canalso be useful for cross-linking anantagomir to another moiety;modifications useful for this include mitomycin C.

Candidate modifications can be evaluated as described below.

The Bases

Adenine, guanine, cytosine and uracil are the most common bases found inRNA. These bases can be modified or replaced to provide RNA's havingimproved properties. For example, nuclease resistantoligoribonucleotides can be prepared with these bases or with syntheticand natural nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine,nubularine, isoguanisine, or tubercidine) and any one of the abovemodifications. Alternatively, substituted or modified analogs of any ofthe above bases, e.g., “unusual bases” and “universal bases” describedherein, can be employed. Examples include without limitation2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo,amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines andguanines, 5-trifluoromethyl and other 5-substituted uracils andcytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, N6, N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-i sopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases. Furtherpurines and pyrimidines include those disclosed in U.S. Pat. No.3,687,808, those disclosed in the Concise Encyclopedia Of PolymerScience And Engineering, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, and those disclosed by Englisch et al., AngewandteChemie, International Edition, 1991, 30, 613.

Candidate modifications can be evaluated as described below.

Evaluation of Candidate Nucleic Acid Agents

One can evaluate a candidate nucleic acid molecule, for a selectedproperty by exposing the molecule or modified molecule and a controlmolecule to the appropriate conditions and evaluating for the presenceof the selected property. For example, resistance to a degradent can beevaluated as follows. A candidate modified nucleic acid molecule can beexposed to degradative conditions, e.g., exposed to a milieu, whichincludes a degradative agent, e.g., a nuclease. For example, one can usea biological sample, e.g., one that is similar to a milieu, which mightbe encountered, in therapeutic use, e.g., blood or a cellular fraction,e.g., a cell-free homogenate or disrupted cells. The candidate andcontrol can then be evaluated for resistance to degradation by any of anumber of approaches. For example, the candidate and control could belabeled, preferably prior to exposure, with, e.g., a radioactive orenzymatic label, or a fluorescent label, such as Cy3 or Cy5. Control andmodified nucleic acid molecules can be incubated with the degradativeagent, and optionally a control, e.g., an inactivated, e.g., heatinactivated, degradative agent. A physical parameter, e.g., size, of themodified and control molecules are then determined. They can bedetermined by a physical method, e.g., by polyacrylamide gelelectrophoresis or a sizing column, to assess whether the molecule hasmaintained its original length, or assessed functionally. Alternatively,Northern blot analysis can be used to assay the length of an unlabeledmodified molecule.

A functional assay can also be used to evaluate the candidate agent. Afunctional assay can be applied initially or after an earliernon-functional assay, (e.g., assay for resistance to degradation) todetermine if the modification alters the ability of the molecule toactivate PRR activity. For example, a cell, e.g., a mammalian cell, suchas a mouse or human cell, can be co-transfected with a plasmid encodinga PRR, and a candidate nucleic acid molecule. In one embodiment, thecandidate oligonucleotide molecule can be assayed for its ability toactivate RIG-I ATPase activity and/or IFN production, as describedelsewhere herein.

RNA may be produced enzymatically or by partial/total organic synthesis,any modified ribonucleotide can be introduced by in vitro enzymatic ororganic synthesis. In one embodiment, the nucleic acid molecule of theinvention is prepared chemically. Methods of synthesizing RNA moleculesare known in the art, in particular, the chemical synthesis methods asdescribed in Verma and Eckstein (1998) Annul Rev. Biochem. 67:99-134.

In one embodiment, the nucleic acid molecule is synthesized either invivo, in situ, or in vitro. Endogenous RNA polymerase of the cell maymediate transcription in vivo or in situ, or cloned RNA polymerase canbe used for transcription in vivo or in vitro. For transcription from atransgene in vivo or an expression construct, a regulatory region (e.g.,promoter, enhancer, silencer, splice donor and acceptor,polyadenylation) may be used to transcribe the RNA molecule. Activity ofthe RNA molecule may be induced by specific transcription in an organ,tissue, or cell type; stimulation of an environmental condition (e.g.,infection, stress, temperature, chemical inducers); and/or engineeringtranscription at a developmental stage or age.

Methods

The invention includes methods of introducing nucleic acids, vectors,and host cells to a subject. Physical methods of introducing nucleicacids include injection of a solution containing the nucleic acidmolecule, bombardment by particles covered by the nucleic acid molecule,soaking the cell or organism in a solution of the nucleic acid molecule,or electroporation of cell membranes in the presence of the nucleic acidmolecule. A viral construct packaged into a viral particle wouldaccomplish both efficient introduction of an expression construct intothe cell and transcription of RNA encoded by the expression construct.Other methods known in the art for introducing nucleic acids to cellsmay be used, such as lipid-mediated carrier transport, chemical-mediatedtransport, such as calcium phosphate, and the like. Thus the nucleicacid may be introduced along with components that perform one or more ofthe following activities: enhance nucleic acid uptake by the cell,stabilize the duplex, or other-wise increase activity of the nucleicacid molecule.

Methods of introducing nucleic acids into a cell are known in the art.The nucleic acid molecule of the invention can be readily introducedinto a host cell, e.g., mammalian, bacterial, yeast, or insect cell byany method in the art. For example, the nucleic acid molecule can betransferred into a host cell by physical, chemical, or biological means.

Physical methods for introducing a nucleic acid into a host cell includecalcium phosphate precipitation, lipofection, particle bombardment,microinjection, electroporation, and the like. Methods for producingcells comprising vectors and/or exogenous nucleic acids are well-knownin the art. See, for example, Sambrook et al. (2001, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

Biological methods for introducing a nucleic acid into a host cellinclude the use of DNA and RNA vectors. Viral vectors, and especiallyretroviral vectors, have become the most widely used method forinserting genes into mammalian, e.g., human cells. Other viral vectorscan be derived from lentivirus, poxviruses, herpes simplex virus I,adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a nucleic acid into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In certain instances, the nucleic acid is delivered via a polymericdelivery vehicle. For example, the nucleic acid molecule may becomplexed with a polymer based micelle, capsule, microparticle,nanoparticle, or the like. The complex may then be contacted to a cellin vivo, in vitro, or ex vivo, thereby introducing the nucleic acidmolecule to the cell. Exemplary polymeric delivery systems are wellknown in the art (see for example U.S. Pat. No. 6,013,240). Polymericdelivery reagents are commercially available, including exemplaryreagents obtainable from Polyplus-transfection Inc (New York, N.Y.).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce the nucleic acid moleculeinto a host cell or otherwise expose a cell to the molecule of thepresent invention, in order to confirm the presence of the nucleic acidin the host cell, a variety of assays may be performed. Such assaysinclude, for example, “molecular biological” assays well known to thoseof skill in the art, such as Southern and Northern blotting, RT-PCR andPCR.

The nucleic acid molecule of the invention may be directly introducedinto the cell (i.e., intracellularly); or introduced extracellularlyinto a cavity, interstitial space, into the circulation of an organism,introduced orally, or may be introduced by bathing a cell or organism ina solution containing the nucleic acid molecule. Vascular orextravascular circulation, the blood or lymph system, and thecerebrospinal fluid are sites where the nucleic acid molecule may beintroduced.

Alternatively, vectors, e.g., transgenes encoding the nucleic acidmolecule of the invention can be engineered into a host cell ortransgenic animal using art recognized techniques.

The present invention provides a method of inducing an IFN response in acell. For example, in certain embodiments, the method induces a type IIFN response. Type I IFNs include, for example IFN-α, IFN-β, IFN-κ,IFN-δ, IFN-ε, IFN-τ, IFN-ω, and IFN-ζ.

The present application provides the in vitro use of the nucleic acidmolecule of the invention. In particular, the present applicationprovides the use of at least one nucleic acid molecule for inducing anIFN response, including for example a type I IFN response, in vitro. Thepresent application also provides the use of at least one nucleic acidmolecule for inducing apoptosis of a tumor cell in vitro.

The present invention provides an in vitro method for stimulating an IFNresponse, including for example a type I IFN response in a cellcomprising contacting a cell with at least one nucleic acid molecule ofthe invention.

The cells may express a PRR endogenously and/or exogenously from anexogenous nucleic acid (RNA or DNA). The exogenous DNA may be a plasmidDNA, a viral vector, or a portion thereof. The exogenous DNA may beintegrated into the genome of the cell or may exist extra-chromosomally.The cells include, but are not limited to, primary immune cells, primarynon-immune cells, and cell lines. Immune cells include, but are notlimited to, peripheral blood mononuclear cells (PBMC), plasmacytoiddendritric cells (PDC), myeloid dendritic cells (MDC), macrophages,monocytes, B cells, natural killer cells, granulocytes, CD4+ T cells,CD8+ T cells, and NKT cells. Non-immune cells include, but are notlimited to, fibroblasts, endothelial cells, epithelial cells, and tumorcells. Cell lines may be derived from immune cells or non-immune cells.

The present invention provides an in vitro method for inducing apoptosisof a tumor cell, comprising contacting a tumor cell with at least onenucleic acid molecule of the invention. The tumor cell may be a primarytumor cell freshly isolated from a vertebrate animal having a tumor or atumor cell line.

In one embodiment, the present invention provides for both prophylacticand therapeutic methods of inducing an IFN response a patient. It isunderstood that “treatment” or “treating” as used herein, is defined asthe application or administration of a therapeutic agent (e.g., anucleic acid molecule) to a patient, or application or administration ofa therapeutic agent to an isolated tissue or cell line from a patient,who has a disease or disorder, a symptom of disease or disorder or apredisposition toward a disease or disorder, with the purpose to cure,heal, alleviate, relieve, alter, remedy, ameliorate, improve or affectthe disease or disorder, the symptoms of the disease or disorder, or thepredisposition toward disease.

In one embodiment, the present application provides the in vivo use ofthe nucleic acid molecule of the invention. In one embodiment, thepresent application provides at least one nucleic acid molecule of theinvention for inducing an IFN response, including for example a type IIFN response, in a vertebrate animal, in particular, a mammal. Thepresent application further provides at least one nucleic acid moleculeof the invention for inducing apoptosis of a tumor cell in a vertebrateanimal, in particular, a mammal. The present application additionallyprovides at least one nucleic acid molecule of the invention forpreventing and/or treating a disease and/or disorder in a vertebrateanimal, in particular, a mammal, in medical and/or veterinary practice.The invention also provides at least one nucleic acid molecule of theinvention for use as a vaccine adjuvant.

In certain embodiments, the composition and method of the invention areused as a research tool. For example, the nucleic acid molecule may beused in vitro or in vivo, to evaluate the effects of increased PRRactivity and/or increased IFN production.

Furthermore, the present application provides the use of at least onenucleic acid molecule of the invention for the preparation of apharmaceutical composition for inducing an IFN response, including forexample a type I IFN response in a vertebrate animal, in particular, amammal. The present application further provides the use of at least onenucleic acid molecule of the invention for the preparation of apharmaceutical composition for inducing apoptosis of a tumor cell in avertebrate animal, in particular, a mammal. The present applicationadditionally provides the use of at least one nucleic acid molecule ofthe invention for the preparation of a pharmaceutical composition forpreventing and/or treating a disease and/or disorder in a vertebrateanimal, in particular, a mammal, in medical and/or veterinary practice.

The present invention encompasses the use of the nucleic acid moleculeto prevent and/or treat any disease, disorder, or condition in whichinducing IFN production would be beneficial. For example, increased IFNproduction, by way of the nucleic acid molecule of the invention, may bebeneficial to prevent or treat a wide variety of disorders, including,but not limited to, bacterial infection, viral infection, parasiticinfection, cancer, immune disorders, respiratory disorders, and thelike.

Infections include, but are not limited to, viral infections, bacterialinfections, anthrax, parasitic infections, fungal infections and prioninfection.

Viral infections include, but are not limited to, infections byhepatitis C, hepatitis B, influenza virus, herpes simplex virus (HSV),human immunodeficiency virus (HIV), respiratory syncytial virus (RSV),vesicular stomatitis virus (VSV), cytomegalovirus (CMV), poliovirus,encephalomyocarditis virus (EMCV), human papillomavirus (HPV) andsmallpox virus. In one embodiment, the infection is an upper respiratorytract infection caused by viruses and/or bacteria, in particular, flu,more specifically, bird flu.

Bacterial infections include, but are not limited to, infections bystreptococci, staphylococci, E. Coli, and pseudomonas. In oneembodiment, the bacterial infection is an intracellular bacterialinfection which is an infection by an intracellular bacterium such asmycobacteria (tuberculosis), chlamydia, mycoplasma, listeria, and anfacultative intracelluar bacterium such as Staphylococcus aureus.

Parasitic infections include, but are not limited to, worm infections,in particular, intestinal worm infection, microeukaryotes, andvector-borne diseases, including for example Leishmaniasis.

In a preferred embodiment, the infection is a viral infection or anintracellular bacterial infection. In a more preferred embodiment, theinfection is a viral infection by hepatitis C, hepatitis B, influenzavirus, RSV, HPV, HSV1, HSV2, and CMV.

Tumors include both benign and malignant tumors (i.e., cancer). Cancersinclude, but are not limited to biliary tract cancer, brain cancer,breast cancer, cervical cancer, choriocarcinoma, colon cancer,endometrial cancer, esophageal cancer, gastric cancer, intraepithelialneoplasm, leukemia, lymphoma, liver cancer, lung cancer, melanoma,myelomas, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer,prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer,thyroid cancer and renal cancer.

In certain embodiments, the cancer is selected from hairy cell leukemia,chronic myelogenous leukemia, cutaneous T-cell leukemia, chronic myeloidleukemia, non-Hodgkin's lymphoma, multiple myeloma, follicular lymphoma,malignant melanoma, squamous cell carcinoma, renal cell carcinoma,prostate carcinoma, bladder cell carcinoma, breast carcinoma, ovariancarcinoma, non-small cell lung cancer, small cell lung cancer,hepatocellular carcinoma, basaliom, colon carcinoma, cervical dysplasia,and Kaposi's sarcoma (AIDS-related and non-AIDS related).

Immune disorders include, but are not limited to, allergies, autoimmunedisorders, and immunodeficiencies.

Allergies include, but are not limited to, respiratory allergies,contact allergies and food allergies.

Autoimmune diseases include, but are not limited to, multiple sclerosis,diabetes mellitus, arthritis (including rheumatoid arthritis, juvenilerheumatoid arthritis, osteoarthritis, psoriatic arthritis),encephalomyelitis, myasthenia gravis, systemic lupus erythematosis,autoimmune thyroiditis, dermatisis (including atopic dermatitis andeczematous dermatitis), psoriasis, Siogren's Syndrome, Crohn's disease,aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerativecolitis, asthma, allergic asthma, cutaneous lupus erythematosus,scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversalreactions, erythema nodosum leprosum, autoimmune uveitis, allergicencephalomyelitis, acute necrotizing hemorrhagic encephalopathy,idiopathic bilateral progressive sensorineural hearing loss, aplasticanemia, pure red cell anemia, idiopathic thrombocytopenia,polychondritis, Wegener's granulomatosis, chronic active hepatitis,Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves'disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, andinterstitial lung fibrosis.

Immunodeficiencies include, but are not limited to, spontaneousimmunodeficiency, acquired immunodeficiency (including AIDS),drug-induced immunodeficiency or immunosuppression (such as that inducedby immunosuppressants used in transplantation and chemotherapeuticagents used for treating cancer), and immunosuppression caused bychronic hemodialysis, trauma or surgical procedures.

Respiratory disorders include, but are not limited to, acute lung injury(ALI), acute respiratory distress syndrome (ARDS), asthma, chronicobstructive pulmonary disease (COPD), obstructive sleep apnea (OSA),idiopathic pulmonary fibrosis (IPF), tuberculosis, pulmonaryhypertension, pleural effusion, and lung cancer.

In certain embodiments, the nucleic acid molecule of the invention isused in combination with one or more pharmaceutically active agents suchas immunostimulatory agents, anti-viral agents, antibiotics, anti-fungalagents, anti-parasitic agents, anti-tumor agents, cytokines, chemokines,growth factors, anti-angiogenic factors, chemotherapeutic agents,antibodies and gene silencing agents. Preferably, the pharmaceuticallyactive agent is selected from the group consisting of animmunostimulatory agent, an anti-bacterial agent, an anti-viral agent,an anti-inflammatory agent and an anti-tumor agent. The more than onepharmaceutically active agents may be of the same or different category.

In one embodiment, the nucleic acid molecule of the invention is used incombination with an antigen, an anti-viral vaccine, an anti-bacterialvaccine, and/or an anti-tumor vaccine, wherein the vaccine can beprophylactic and/or therapeutic. The nucleic acid molecule can serve asan adjuvant.

In another embodiment, the nucleic acid is used in combination withretinoic acid and/or type I IFN (IFN-α and/or IFN-β). Without beingbound by any theory, retinoid acid, IFN-α and/or IFN-β are capable ofsensitizing cells for IFN-β production, possibly through theupregulation of PRR expression.

In other embodiments, the nucleic acid molecule of the invention is foruse in combination with one or more prophylactic and/or therapeutictreatments of diseases and/or disorders such as infection, tumor, andimmune disorders. The treatments may be pharmacological and/or physical(e.g., surgery, radiation).

Vertebrate animals include, but are not limited to, fish, amphibians,birds, and mammals. Mammals include, but are not limited to, rats, mice,cats, dogs, horses, sheep, cattle, cows, pigs, rabbits, non-humanprimates, and humans. In a preferred embodiment, the mammal is human.

In one embodiment, the nucleic acid molecule of the invention is used incombination with an anti-viral vaccine, wherein the vaccine can beprophylactic and/or therapeutic.

Administration/Dosing

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations may be administered to the subjecteither prior to or after a diagnosis of disease. Further, severaldivided dosages, as well as staggered dosages may be administered dailyor sequentially, or the dose may be continuously infused, or may be abolus injection. Further, the dosages of the therapeutic formulationsmay be proportionally increased or decreased as indicated by theexigencies of the therapeutic or prophylactic situation.

Administration of the compositions of the present invention to asubject, preferably a mammal, more preferably a human, may be carriedout using known procedures, at dosages and for periods of time effectiveto prevent or treat disease. An effective amount of the therapeuticcompound necessary to achieve a therapeutic effect may vary according tofactors such as the activity of the particular compound employed; thetime of administration; the rate of excretion of the compound; theduration of the treatment; other drugs, compounds or materials used incombination with the compound; the state of the disease or disorder,age, sex, weight, condition, general health and prior medical history ofthe subject being treated, and like factors well-known in the medicalarts. Dosage regimens may be adjusted to provide the optimum therapeuticresponse. For example, several divided doses may be administered dailyor the dose may be proportionally reduced as indicated by the exigenciesof the therapeutic situation. A non-limiting example of an effectivedose range for a therapeutic compound of the invention is from about 1and 5,000 mg/kg of body weight/per day. One of ordinary skill in the artwould be able to study the relevant factors and make the determinationregarding the effective amount of the therapeutic compound without undueexperimentation.

The compound may be administered to a subject as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. It is understood that the amount of compound dosed per day may beadministered, in non-limiting examples, every day, every other day,every 2 days, every 3 days, every 4 days, or every 5 days. For example,with every other day administration, a 5 mg per day dose may beinitiated on Monday with a first subsequent 5 mg per day doseadministered on Wednesday, a second subsequent 5 mg per day doseadministered on Friday, and so on. The frequency of the dose will bereadily apparent to the skilled artisan and will depend upon any numberof factors, such as, but not limited to, the type and severity of thedisease being treated, the type and age of the animal, etc.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular subject, composition, and mode ofadministration, without being toxic to the subject.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding/formulating such a therapeutic compound for thetreatment of a disease in a subject.

In one embodiment, the compositions of the invention are administered tothe subject in dosages that range from one to five times per day ormore. In another embodiment, the compositions of the invention areadministered to the subject in range of dosages that include, but arenot limited to, once every day, every two, days, every three days toonce a week, and once every two weeks. It will be readily apparent toone skilled in the art that the frequency of administration of thevarious combination compositions of the invention will vary from subjectto subject depending on many factors including, but not limited to, age,disease or disorder to be treated, gender, overall health, and otherfactors. Thus, the invention should not be construed to be limited toany particular dosage regime and the precise dosage and composition tobe administered to any subject will be determined by the attendingphysical taking all other factors about the subject into account.

Compounds of the invention for administration may be in the range offrom about 1 mg to about 10,000 mg, about 20 mg to about 9,500 mg, about40 mg to about 9,000 mg, about 75 mg to about 8,500 mg, about 150 mg toabout 7,500 mg, about 200 mg to about 7,000 mg, about 3050 mg to about6,000 mg, about 500 mg to about 5,000 mg, about 750 mg to about 4,000mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg toabout 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about400 mg to about 500 mg, and any and all whole or partial incrementstherebetween.

In some embodiments, the dose of a compound of the invention is fromabout 1 mg and about 2,500 mg. In some embodiments, a dose of a compoundof the invention used in compositions described herein is less thanabout 10,000 mg, or less than about 8,000 mg, or less than about 6,000mg, or less than about 5,000 mg, or less than about 3,000 mg, or lessthan about 2,000 mg, or less than about 1,000 mg, or less than about 500mg, or less than about 200 mg, or less than about 50 mg. Similarly, insome embodiments, a dose of a second compound (i.e., a drug used fortreating the same or another disease as that treated by the compositionsof the invention) as described herein is less than about 1,000 mg, orless than about 800 mg, or less than about 600 mg, or less than about500 mg, or less than about 400 mg, or less than about 300 mg, or lessthan about 200 mg, or less than about 100 mg, or less than about 50 mg,or less than about 40 mg, or less than about 30 mg, or less than about25 mg, or less than about 20 mg, or less than about 15 mg, or less thanabout 10 mg, or less than about 5 mg, or less than about 2 mg, or lessthan about 1 mg, or less than about 0.5 mg, and any and all whole orpartial increments thereof.

In one embodiment, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound or conjugate of theinvention, alone or in combination with a second pharmaceutical agent;and instructions for using the compound or conjugate to treat, prevent,or reduce one or more symptoms of a disease in a subject.

The term “container” includes any receptacle for holding thepharmaceutical composition. For example, in one embodiment, thecontainer is the packaging that contains the pharmaceutical composition.In other embodiments, the container is not the packaging that containsthe pharmaceutical composition, i.e., the container is a receptacle,such as a box or vial that contains the packaged pharmaceuticalcomposition or unpackaged pharmaceutical composition and theinstructions for use of the pharmaceutical composition. Moreover,packaging techniques are well known in the art. It should be understoodthat the instructions for use of the pharmaceutical composition may becontained on the packaging containing the pharmaceutical composition,and as such the instructions form an increased functional relationshipto the packaged product. However, it should be understood that theinstructions may contain information pertaining to the compound'sability to perform its intended function, e.g., treating or preventing adisease in a subject, or delivering an imaging or diagnostic agent to asubject.

Pharmaceutical Compositions

The present invention provides a pharmaceutical composition comprisingat least one nucleic acid molecule of the present invention and apharmaceutically acceptable carrier. The formulations of thepharmaceutical compositions described herein may be prepared by anymethod known or hereafter developed in the art of pharmacology. Ingeneral, such preparatory methods include the step of bringing theactive ingredient into association with a carrier or one or more otheraccessory ingredients, and then, if necessary or desirable, shaping orpackaging the product into a desired single- or multi-dose unit.

Although the description of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as non-human primates, cattle, pigs, horses,sheep, cats, and dogs.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary,intranasal, buccal, or another route of administration. Othercontemplated formulations include projected nanoparticles, liposomalpreparations, resealed erythrocytes containing the active ingredient,and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents. Other active agents useful in thepresent invention include anti-inflammatories, includingcorticosteroids, and immunosuppressants, chemotherapeutic agents,antibiotics, antivirals, antifungals, and the like.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology,using for example proteins equipped with pH sensitive domains orprotease-cleavable fragments. In some cases, the dosage forms to be usedcan be provided as slow or controlled-release of one or more activeingredients therein using, for example, hydropropylmethyl cellulose,other polymer matrices, gels, permeable membranes, osmotic systems,multilayer coatings, micro-particles, liposomes, or microspheres or acombination thereof to provide the desired release profile in varyingproportions. Suitable controlled-release formulations known to those ofordinary skill in the art, including those described herein, can bereadily selected for use with the pharmaceutical compositions of theinvention. Thus, single unit dosage forms suitable for oraladministration, such as tablets, capsules, gel-caps, and caplets, whichare adapted for controlled-release are encompassed by the presentinvention.

Most controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased subject compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood level of the drug, andthus can affect the occurrence of side effects.

Most controlled-release formulations are designed to initially releasean amount of drug that promptly produces the desired therapeutic effect,and gradually and continually release of other amounts of drug tomaintain this level of therapeutic effect over an extended period oftime. In order to maintain this constant level of drug in the body, thedrug must be released from the dosage form at a rate that will replacethe amount of drug being metabolized and excreted from the body.

Controlled-release of an active ingredient can be stimulated by variousinducers, for example pH, temperature, enzymes, water or otherphysiological conditions or compounds. The term “controlled-releasecomponent” in the context of the present invention is defined herein asa compound or compounds, including, but not limited to, polymers,polymer matrices, gels, permeable membranes, liposomes, or microspheresor a combination thereof that facilitates the controlled-release of theactive ingredient.

In certain embodiments, the formulations of the present invention maybe, but are not limited to, short-term, rapid-offset, as well ascontrolled, for example, sustained release, delayed release andpulsatile release formulations.

The term sustained release is used in its conventional sense to refer toa drug formulation that provides for gradual release of a drug over anextended period of time, and that may, although not necessarily, resultin substantially constant blood levels of a drug over an extended timeperiod. The period of time may be as long as a month or more and shouldbe a release that is longer that the same amount of agent administeredin bolus form.

For sustained release, the compounds may be formulated with a suitablepolymer or hydrophobic material that provides sustained releaseproperties to the compounds. As such, the compounds for use the methodof the invention may be administered in the form of microparticles, forexample, by injection or in the form of wafers or discs by implantation.

In a preferred embodiment of the invention, the compounds of theinvention are administered to a subject, alone or in combination withanother pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that mat,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours.

The term pulsatile release is used herein in its conventional sense torefer to a drug formulation that provides release of the drug in such away as to produce pulsed plasma profiles of the drug after drugadministration.

The term immediate release is used in its conventional sense to refer toa drug formulation that provides for release of the drug immediatelyafter drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Remington's PharmaceuticalSciences (1985, Genaro, ed., Mack Publishing Co., Easton, Pa.), which isincorporated herein by reference.

The composition of the invention may comprise a preservative from about0.005% to 2.0% by total weight of the composition. The preservative isused to prevent spoilage in the case of exposure to contaminants in theenvironment. Examples of preservatives useful in accordance with theinvention included but are not limited to those selected from the groupconsisting of benzyl alcohol, sorbic acid, parabens, imidurea andcombinations thereof. A particularly preferred preservative is acombination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5%sorbic acid.

The composition preferably includes an anti-oxidant and a chelatingagent that inhibits the degradation of the compound. Preferredantioxidants for some compounds are BHT, BHA, alpha-tocopherol andascorbic acid in the preferred range of about 0.01% to 0.3% and morepreferably BHT in the range of 0.03% to 0.1% by weight by total weightof the composition. Preferably, the chelating agent is present in anamount of from 0.01% to 0.5% by weight by total weight of thecomposition. Particularly preferred chelating agents include edetatesalts (e.g. disodium edetate) and citric acid in the weight range ofabout 0.01% to 0.20% and more preferably in the range of 0.02% to 0.10%by weight by total weight of the composition. The chelating agent isuseful for chelating metal ions in the composition that may bedetrimental to the shelf life of the formulation. While BHT and disodiumedetate are the particularly preferred antioxidant and chelating agentrespectively for some compounds, other suitable and equivalentantioxidants and chelating agents may be substituted therefore as wouldbe known to those skilled in the art.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water, and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.,polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin, and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. As used herein, an “oily” liquidis one which comprises a carbon-containing liquid molecule and whichexhibits a less polar character than water. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water, and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e., such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Routes of administration of any of the compositions of the inventioninclude oral, nasal, rectal, parenteral, sublingual, transdermal,transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral,vaginal (e.g., trans- and perivaginally), (intra)nasal, and(trans)rectal), intravesical, intrapulmonary, intraduodenal,intragastrical, intrathecal, subcutaneous, intramuscular, intradermal,intra-arterial, intravenous, intrabronchial, inhalation, and topicaladministration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

For oral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules, caplets and gelcaps. Otherformulations suitable for oral administration include, but are notlimited to, a powdered or granular formulation, an aqueous or oilysuspension, an aqueous or oily solution, a paste, a gel, toothpaste, amouthwash, a coating, an oral rinse, or an emulsion. The compositionsintended for oral use may be prepared according to any method known inthe art and such compositions may contain one or more agents selectedfrom the group consisting of inert, non-toxic pharmaceuticallyexcipients that are suitable for the manufacture of tablets. Suchexcipients include, for example an inert diluent such as lactose;granulating and disintegrating agents such as cornstarch; binding agentssuch as starch; and lubricating agents such as magnesium stearate.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmoticallycontrolled release tablets. Tablets may further comprise a sweeteningagent, a flavoring agent, a coloring agent, a preservative, or somecombination of these in order to provide for pharmaceutically elegantand palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

For oral administration, the compositions of the invention may be in theform of tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents; fillers;lubricants; disintegrates; or wetting agents. If desired, the tabletsmay be coated using suitable methods and coating materials such asOPADRY™ film coating systems available from Colorcon, West Point, Pa.(e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, AqueousEnteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400).

Liquid preparation for oral administration may be in the form ofsolutions, syrups or suspensions. The liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifying agent (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily estersor ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid). Liquid formulations of a pharmaceuticalcomposition of the invention which are suitable for oral administrationmay be prepared, packaged, and sold either in liquid form or in the formof a dry product intended for reconstitution with water or anothersuitable vehicle prior to use.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface-active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Granulating techniques are well known in the pharmaceutical art formodifying starting powders or other particulate materials of an activeingredient. The powders are typically mixed with a binder material intolarger permanent free-flowing agglomerates or granules referred to as a“granulation.” For example, solvent-using “wet” granulation processesare generally characterized in that the powders are combined with abinder material and moistened with water or an organic solvent underconditions resulting in the formation of a wet granulated mass fromwhich the solvent must then be evaporated.

Melt granulation generally consists in the use of materials that aresolid or semi-solid at room temperature (i.e. having a relatively lowsoftening or melting point range) to promote granulation of powdered orother materials, essentially in the absence of added water or otherliquid solvents. The low melting solids, when heated to a temperature inthe melting point range, liquefy to act as a binder or granulatingmedium. The liquefied solid spreads itself over the surface of powderedmaterials with which it is contacted, and on cooling, forms a solidgranulated mass in which the initial materials are bound together. Theresulting melt granulation may then be provided to a tablet press or beencapsulated for preparing the oral dosage form. Melt granulationimproves the dissolution rate and bioavailability of an active (i.e.drug) by forming a solid dispersion or solid solution.

U.S. Pat. No. 5,169,645 discloses directly compressible wax-containinggranules having improved flow properties. The granules are obtained whenwaxes are admixed in the melt with certain flow improving additives,followed by cooling and granulation of the admixture. In certainembodiments, only the wax itself melts in the melt combination of thewax(es) and additives(s), and in other cases both the wax(es) and theadditives(s) will melt.

The present invention also includes a multi-layer tablet comprising alayer providing for the delayed release of one or more compounds of theinvention, and a further layer providing for the immediate release of amedication for treatment of a disease. Using a wax/pH-sensitive polymermix, a gastric insoluble composition may be obtained in which the activeingredient is entrapped, ensuring its delayed release.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, intraocular,intravitreal, subcutaneous, intraperitoneal, intramuscular, intrasternalinjection, intratumoral, and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g. sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

An obstacle for topical administration of pharmaceuticals is the stratumcorneum layer of the epidermis. The stratum corneum is a highlyresistant layer comprised of protein, cholesterol, sphingolipids, freefatty acids and various other lipids, and includes cornified and livingcells. One of the factors that limit the penetration rate (flux) of acompound through the stratum corneum is the amount of the activesubstance that can be loaded or applied onto the skin surface. Thegreater the amount of active substance which is applied per unit of areaof the skin, the greater the concentration gradient between the skinsurface and the lower layers of the skin, and in turn the greater thediffusion force of the active substance through the skin. Therefore, aformulation containing a greater concentration of the active substanceis more likely to result in penetration of the active substance throughthe skin, and more of it, and at a more consistent rate, than aformulation having a lesser concentration, all other things being equal.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions. Topicallyadministrable formulations may, for example, comprise from about 1% toabout 10% (w/w) active ingredient, although the concentration of theactive ingredient may be as high as the solubility limit of the activeingredient in the solvent. Formulations for topical administration mayfurther comprise one or more of the additional ingredients describedherein.

Enhancers of permeation may be used. These materials increase the rateof penetration of drugs across the skin. Typical enhancers in the artinclude ethanol, glycerol monolaurate, PGML (polyethylene glycolmonolaurate), dimethylsulfoxide, and the like. Other enhancers includeoleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylicacids, dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone.

One acceptable vehicle for topical delivery of some of the compositionsof the invention may contain liposomes. The composition of the liposomesand their use are known in the art (for example, see U.S. Pat. No.6,323,219).

In alternative embodiments, the topically active pharmaceuticalcomposition may be optionally combined with other ingredients such asadjuvants, anti-oxidants, chelating agents, surfactants, foaming agents,wetting agents, emulsifying agents, viscosifiers, buffering agents,preservatives, and the like. In another embodiment, a permeation orpenetration enhancer is included in the composition and is effective inimproving the percutaneous penetration of the active ingredient into andthrough the stratum corneum with respect to a composition lacking thepermeation enhancer. Various permeation enhancers, including oleic acid,oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids,dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone, are known tothose of skill in the art. In another aspect, the composition mayfurther comprise a hydrotropic agent, which functions to increasedisorder in the structure of the stratum corneum, and thus allowsincreased transport across the stratum corneum. Various hydrotropicagents, such as isopropyl alcohol, propylene glycol, or sodium xylenesulfonate, are known to those of skill in the art.

The topically active pharmaceutical composition should be applied in anamount effective to affect desired changes. As used herein “amounteffective” shall mean an amount sufficient to cover the region of skinsurface where a change is desired. An active compound should be presentin the amount of from about 0.0001% to about 15% by weight volume of thecomposition. More preferable, it should be present in an amount fromabout 0.0005% to about 5% of the composition; most preferably, it shouldbe present in an amount of from about 0.001% to about 1% of thecomposition. Such compounds may be synthetically- or naturally derived.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations may be made by combining the active ingredientwith a non-irritating pharmaceutically acceptable excipient which issolid at ordinary room temperature (i.e., about 20° C.) and which isliquid at the rectal temperature of the subject (i.e., about 37° C. in ahealthy human). Suitable pharmaceutically acceptable excipients include,but are not limited to, cocoa butter, polyethylene glycols, and variousglycerides. Suppository formulations may further comprise variousadditional ingredients including, but not limited to, antioxidants, andpreservatives.

Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants, and preservatives.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for vaginal administration. Withrespect to the vaginal or perivaginal administration of the compounds ofthe invention, dosage forms may include vaginal suppositories, creams,ointments, liquid formulations, pessaries, tampons, gels, pastes, foamsor sprays. The suppository, solution, cream, ointment, liquidformulation, pessary, tampon, gel, paste, foam or spray for vaginal orperivaginal delivery comprises a therapeutically effective amount of theselected active agent and one or more conventional nontoxic carrierssuitable for vaginal or perivaginal drug administration. The vaginal orperivaginal forms of the present invention may be manufactured usingconventional processes as disclosed in Remington: The Science andPractice of Pharmacy, supra (see also drug formulations as adapted inU.S. Pat. Nos. 6,515,198; 6,500,822; 6,417,186; 6,416,779; 6,376,500;6,355,641; 6,258,819; 6,172,062; and 6,086,909). The vaginal orperivaginal dosage unit may be fabricated to disintegrate rapidly orover a period of several hours. The time period for completedisintegration may be in the range of from about 10 minutes to about 6hours, e.g., less than about 3 hours.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e., such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Douche preparations or solutions for vaginal irrigation may be made bycombining the active ingredient with a pharmaceutically acceptableliquid carrier. As is well known in the art, douche preparations may beadministered using, and may be packaged within, a delivery deviceadapted to the vaginal anatomy of the subject.

Douche preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants, antibiotics, antifungalagents, and preservatives.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers, and preferably from about 1 toabout 6 nanometers.

Such compositions are conveniently in the form of dry powders foradministration using a device comprising a dry powder reservoir to whicha stream of propellant may be directed to disperse the powder or using aself-propelling solvent/powder-dispensing container such as a devicecomprising the active ingredient dissolved or suspended in a low-boilingpropellant in a sealed container. Preferably, such powders compriseparticles wherein at least 98% of the particles by weight have adiameter greater than 0.5 nanometers and at least 95% of the particlesby number have a diameter less than 7 nanometers. More preferably, atleast 95% of the particles by weight have a diameter greater than 1nanometer and at least 90% of the particles by number have a diameterless than 6 nanometers. Dry powder compositions preferably include asolid fine powder diluent such as sugar and are conveniently provided ina unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 micrometers. Such a formulation is administered inthe manner in which snuff is taken i.e. by rapid inhalation through thenasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, 0.1 to 20% (w/w)active ingredient, the balance comprising an orally dissolvable ordegradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration may comprise a powder or an aerosolized oratomized solution or suspension comprising the active ingredient. Suchpowdered, aerosolized, or aerosolized formulations, when dispersed,preferably have an average particle or droplet size in the range fromabout 0.1 to about 200 nanometers, and may further comprise one or moreof the additional ingredients described herein.

Diagnosing and Screening Assay

In one embodiment, the present invention provides compositions andmethods for detecting a PRR in a biological sample and diagnosing adisease or disorder associated with a PRR in a subject. In oneembodiment, the present invention provides compositions and methods fordiagnosing a disease or disorder associated with an IFN response in asubject.

In one embodiment, the method comprises assessing the presence and/oractivity of a PRR in a subject using a nucleic acid molecule of theinvention. For example, a cell or biological sample is isolated from thesubject and the cell or biological sample is contacted with a nucleicacid molecule of the invention to determine whether the cell orbiological sample is able to induce an IFN response.

In one embodiment, the assay comprises using a nucleic acid molecule ofthe invention to determine whether a cell or a biological samplecomprising a cell exhibits PRR activity. For example, the cell orbiological sample is contacted with a nucleic acid molecule of theinvention to determine whether the cell or biological sample is able toinduce an IFN response. Without wishing to be bound by any particulartheory, a cell or biological sample that induces an IFN response in thepresence of a nucleic acid molecule of the invention compared to a cellor biological sample that does not induce an IFN response means that thecell or biological sample that induces an IFN response comprises a PRR.

In one aspect, the present invention is directed to a screening assay toidentify compounds that stimulate or inhibit PRR activity. In anotheraspect, the present invention is directed to a screening assay toidentify compounds that induce or inhibit an IFN response.

In one embodiment, the invention provides a method of screening alibrary of agents to identify an agent that induces or inhibits an IFNresponse. For example, the method comprises contacting a cell orbiological sample with a nucleic acid molecule of the invention in thepresence or absence of a test compound. Without wishing to be bound byany particular theory, a cell or biological sample that induces an IFNresponse or increases an IFN response in the presence of a nucleicmolecule of the invention and the test agent identifies the test agentas one that induces IFN response. For example, in one embodiment, thelevel of IFN response in the presence of a nucleic acid molecule of theinvention is a baseline level for an IFN response. An agent that inducesan IFN response is identified when the level of IFN response isincreased when the cell or biological sample is combined with thenucleic acid molecule of the invention and the test agent. On the otherhand, an agent that inhibits an IFN response is identified when thelevel of IFN response is decreased when the cell or biological sample iscombined with the nucleic acid molecule of the invention and the testagent.

The test agents can be obtained using any of the numerous approaches incombinatorial-library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the“one-bead one-compound” library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam et al., 1997, Anticancer Drug Des. 12:45).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example, in: DeWitt et al., 1993, Proc. Natl.Acad. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al.,1993, Science 261:1303; Carrell et al., 1994, Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al., 1994, J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten,1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage(Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science249:404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; and Ladnersupra).

In situations where “high-throughput” modalities are preferred, it istypical that new chemical entities with useful properties are generatedby identifying a chemical compound (called a “lead compound”) with somedesirable property or activity, creating variants of the lead compound,and evaluating the property and activity of those variant compounds. Thecurrent trend is to shorten the time scale for all aspects of drugdiscovery.

In one embodiment, high throughput screening methods involve providing alibrary containing a large number of compounds (candidate compounds)potentially having the desired activity. Such “combinatorial chemicallibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

Kits

The invention also provides kits stimulating PRR activity and inducingan IFN response, as elsewhere described herein. In one embodiment, thekit includes a composition comprising a nucleic acid molecule, aselsewhere described herein, and instructions for its use. Theinstructions will generally include information about the use of thecompositions in the kit for the stimulation of PRR activity. Theinstructions may be printed directly on a container inside the kit (whenpresent), or as a label applied to the container, or as a separatesheet, pamphlet, card, or folder supplied in or with the container.

The invention also provides kits for the treatment or prevention of adisease, disorder, or condition in which IFN production would bebeneficial. In one embodiment, the kit includes a composition (e.g. apharmaceutical composition) comprising a nucleic acid molecule, aselsewhere described herein, and instructions for its use. Theinstructions will generally include information about the use of thecompositions in the kit for the treatment or prevention of a disease ordisorder or symptoms thereof. The instructions may be printed directlyon a container inside the kit (when present), or as a label applied tothe container, or as a separate sheet, pamphlet, card, or foldersupplied in or with the container.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless so specified. Thus, the invention should in no way be construedas being limited to the following examples, but rather, should beconstrued to encompass any and all variations which become evident as aresult of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1: Defining the Functional Determinants for RNA Surveillance byRIG-I

Retinoic acid inducible gene-I (RIG-I) is an intracellular RNA sensorthat engages the innate immune machinery in response to infection by avariety of RNA viruses. The pathogen associated molecular pattern (PAMP)for RIG-I is generally defined as duplex RNA containing a 5′triphosphatemoiety. The results presented herein demonstrate an additional twodistinct conformations of a RIG-I: dsRNA complex that illustrate thestructural dynamics of RNA duplex recognition and its relevance to thecatalytic ATPase activity of RIG-I. Reported herein, are the crystalstructure of distinct conformations of a RIG-I:dsRNA complex, whichshows that HEL2i-mediated scanning allows RIG-I to sense the length ofRNA targets. While the Hel1-RNA-CTD form a rigid sandwich-like fold, theHel2i domain of RIG-I exhibits a high degree of flexibility in surveyingthe substrate, making contacts with six to ten base pairs of the RNA. Toelucidate the significance of this scanning mechanism, the ability ofRNA duplexes to stimulate the ATP hydrolysis activity of RIG-I andelicit an interferon response was measured. Short RNA hairpins andpalindromic duplexes of lengths between 8 and 30 base pairs and with 5′ends of either a hydroxyl group or triphosphate were tested. The resultspresented herein provide biophysical and in vivo evidence that RIG-Iactivity is stimulated exclusively via interaction with the 5′ ends ofduplex substrates, whereas interactions with internal “stem” regions ofthese substrates are likely non-productive. The data indicate RIG-Isurveys and recognizes 5′ ends of dsRNA as a monomer without RNA-inducedoligomerization. These results reveal that the minimal functional unitof the RIG-I:RNA complex is a monomer that binds at the terminus of aduplex RNA substrate. This behaviour is markedly different from theRIG-I paralog melanoma differentiation-associated gene 5 (MDA5), whichforms cooperative filaments.

The materials and methods employed in these experiments are nowdescribed.

Cloning, Expression, and Purification

Purification of the full-length human RIG-I and the N-terminal CARDs(residues 1-229) deletion construct was described previously (Luo etal., 2011, Cell 147:409-422; Luo et al., 2012b, Structure,20:1983-1988). Briefly, the constructs were cloned into the pET-SUMOvector (Invitrogen) and transformed into Rosetta II(DE3) E. coli cells(Novagen). The proteins were expressed in LB media upon the addition of0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and grown at 18° C.overnight for 20 hours. The cells were then lysed with a microfluidizerat 15,000 psi, clarified by centrifugation, and purified by batchbinding with Ni-NTA beads (Qiagen). After collection and elution fromBiorad polyprep columns, the RIG-I constructs were concentrated on aHiTrap Heparin HP column (GE Healthcare) and gel filtered over a HiPrep16/60 Superdex 200 column (GE Healthcare) in buffer containing 25 mMHepes, pH 7.4, 150 mM NaCl, 5% glycerol, 5 mM β-ME. RIG-I preparationswere concentrated to between 5-10 mg/mL with a 50 k MW cutoff Amiconcentrifugal concentrator (Millipore), and concentrations were determinedspectrophotometrically, using the extinction coefficients of c=99,700M-1 cm-1 for full length RIG-I and an c=60,280 M-1 cm-1 for the RIG-I(ΔCARDs:1-229)N-terminal deletion construct. The extinction coefficientswere calculated theoretically from the RIG-I sequence and guanidiniumchloride denaturation of protein preparations. The RIG-I preparationswere flash frozen with liquid nitrogen and stored at −80° C.

RNA Synthesis and Transcription

The 5′OH ‘GC’ palindromic RNA duplexes were made by RNA synthesis(Sigma). The 5′ppp ‘GC’ palindromic RNA duplexes were made by in vitrorunoff transcription using DNA templates (SIGMA) with a 2′O-methylmodification on the penultimate nucleotide of the template strand (Kaoet al., 1999, RNA, 5: 1268-72). The LMW poly I:C was ordered fromInvivogen. The 2′O-methyl modification on the penultimate nucleotide ofthe template strand prevents T7 terminal transferase activity aspreviously described (Kao et al., 1999, RNA, 5: 1268-72). Incorporationof 2′-OMe modifications within the DNA template prevents addition of +1and +2 nucleotides by T7 RNA polymerase and results in transcription ofRNA molecules with defined, uniform 3′-ends, which is obviouslyessential for studies of RIG-I binding and stimulation. All synthesizedand transcribed RNA constructs were purified on 20% denaturingpolyacrylamide gels. LMW polyI:C (Invivogen) was dissolved in buffercontaining Hepes (pH 7.4), 150 mM NaCl, 5% glycerol, 5 mM BME to a finalconcentration of 10 mg/ml. 500 μl of this solution was loaded on ananalytical 10.300 Superdex 200 Column (GE) and eluted at 0.25 ml/minwhile collecting 1 ml fractions. Concentrations were determinedspectrophotometrically.

All hairpin RNAs were purified by 8M urea PAGE. After gel extraction,the re-annealing step was performed at low RNA concentrations by heatingthe RNA at 96° C. for 2 mins and rapidly cooling on ice. It is notablethat these hairpins are stabilized by a terminal UUCG tetraloop, whichis known to promote exclusive hairpin formation by short duplexes,including those as short as four base-pairs (Cheong et al, 1990, Nature,346: 680-682; Nozinovic et al, 2010, Nucleic Acids Res, 38: 683-694).

Crystallization, Data Collection, Structure Determination andRefinement.

The crystallization and data collection of RIG-I (ΔCARDs:1-229) binaryand ternary complexes were performed as described previously, withmodifications (Luo et al., 2011, Cell 147:409-422). Structures weredetermined by molecular replacement using pdb:2ykg as a model. Briefly,the RIG-I (ΔCARDs:1-229) complex with 5′OH-GC10 duplex was preassembledby incubating at a protein:RNA molar ratio of 1:1.5 on ice for 1 hourand then purified with a HiPrep 16/60 Superdex 200 column (GEHealthcare). The crystals of the binary complex of RIG-I(ΔCARDs:1-229):5′OH-GC10 were grown at 13° C. by mixing equal volumes ofprecipitating solution (0.1 M Bicine, pH 9.0, 22.5% polyethylene glycol6,000) and RIG-I (ΔCARDs:1-229): 5′OH-GC10 complex (2-3 mg ml⁻¹) usingthe sitting drop method. The crystals grew into needle clusters within aweek and were harvested within two weeks. The crystals were soaked in acryoprotecting solution containing 0.1 M Bicine, pH 9.0, 30%polyethylene glycol 6,000 for 12 hours before being flash frozen withliquid nitrogen. To grow the crystals of the ternary complex of RIG-I(ΔCARDs:1-229):5′OH-GC10:ADP-Mg²⁺, the binary complex of RIG-I(ΔCARDs:1-229):5′OH-GC10 was first incubated with 2.5 mM ADP and 2.5 mMMgCl₂ at 2-3 mg ml⁻¹ for half an hour to one hour on ice, mixed withequal volumes of precipitating solution (0.1 M Bicine, pH 9.0, 26-28%polyethylene glycol 6,000) and then grown at 13° C. Crystals also grewinto needle clusters within three days and were harvested within twoweeks. Crystals were soaked in a cryoprotecting solution containing 0.1M Bicine, pH 9.0, 30% polyethylene glycol 6,000 briefly before beingflash frozen with liquid nitrogen. Diffraction intensities were recordedat NE-CAT beamline ID-24 at the Advanced Photon Source (Argonne NationalLaboratory, Argonne, Ill.). Integration, scaling and merging of theintensities were carried out by using the programs XDS (Kabsch, 2010,Acta Crystallogr D Biol Crystallogr 66:125-132) and SCALA (Evans, 2006,Acta Crystallogr D Biol Crystallogr 62:72-82).

Initial attempts to use the structure of RIG-I (ΔCARDs:1-229): 5′OH-GC10(PDB: 2ykg) as search model for molecular replacement were notsuccessful. Rather, successful phasing was accomplished throughmolecular replacement by using the subgroups (HEL1: aa 236-455,HEL2-HEL2i: aa 456-793, CTD: aa 794-925, and dsRNA) of RIG-I(ΔCARDs:1-229): 5′OH-GC10 (PDB: 2ykg) as search models in Phaser (McCoy,2007, Acta Crystallogr D Biol Crystallogr 63:32-41). Refinement cycleswere carried out using Phenix Refine (Adams et al., 2010, ActaCrystallogr D Biol Crystallogr 66:213-221) and REFMACS (Murshudov etal., 1997, Acta Crystallogr D Biol Crystallogr 53:240-255) with four TLS(translation, liberation, screw-rotation displacement) groups (HEL1: aa236-455, HEL2-HEL2i: aa 456-793, CTD: aa 794-925, and dsRNA). Refinementcycles were interspersed with model rebuilding using Coot (Emsley andCowtan, 2004, Acta Crystallogr D Biol Crystallogr 60:2126-2132). Thequality of the structures was analyzed with PROCHECK (Laskowski et al.,1993, J Appl Cryst 26:283-291). A summary of the data collection andstructure refinement statistics is given in Table 1. During thecrystallographic studies, it was noticed that crystals with RIG-I:dsRNAcaptured in the conformation 1, the binary complex of RIG-I and5′OH-GC10, is always associated with the longest c axis (225.1 Å) of theunit cells (conformation 2, 219.8 Å and conformation 3, 207.8 Å).Figures were prepared by using the program Pymol (DeLano, 2002, ThePyMOL User's Manual: DeLano Scientific, Palo Alto, Calif., USA).

TABLE 1 Crystallographic and structure refinement statistics. RIG-I(ΔCARDs 1-229): RIG-I (ΔCARDs 1-229): RIG-I (ΔCARDs 1-229): GC10 GC10:SO

GC10: ADP-MG Structure (Conformation 1) (Conformation 2) ^(b)(Conformation 3) Data collection Space group P2₁2₁2₁ P2

2

2

P2₁2₁2₁ Cell dimensions (Å) 48.5, 78.0, 225.1 47.6, 76.2, 219.8 48.3,76.1, 207.

Resolution (Å)  48.5-2.8 (2.9-2.8) ^(a) 47.6-2.5 (2.6-2.5)  48.3-2.5(2.6-2.5)  R merge (%)  6.4 (70.6)  7.5 (62.3)  6.4 (57.3) 1/

16.2 (1.8)  14.5 (1.5)  11.4 (1.9)  Completeness (%) 99.3 (98.6) 93.8(58.8) 98.1 (98.9) Redundancy 5.0 (4.8) 5.0 (2.2) 3.4 (3.1) RefinementResolution (Å) 25.0-2.8 45.0-2.5 25.0-2.5 R work/R free (%) 22.2/27.922.4/27.5 22.9/28.8 No. atoms 5,380 5,517 5,369 Protein 4,947 4,9854,857 RNA/ADP-Mg²⁺ 424 424 424/28 Water 9 99 60 B-factors (Å²) 74.7 57.966.2 Protein 75.1 58.1 66.4 Ligand 71.7 65.7 70.2 Solvent 74.6 48.2 51.9R

ch

dran analysis Favored 86.3 94.2 89.1 Additionally allowed 12.8 4.8 10.4Outliers 0.9 1.0 0.5 R.m.s. deviations Bond lengths (Å) 0.099 0.0860.097 Bond angles (°) 1.6 1.3 1.5 PDB ID 3zd6 2ykg 3zd7 ^(a) Statisticsfor the highest resolution shell is shown in parenthesis ^(b) Referencedata taken from RCSB Protein Data Bank (ID: 2YKG) (Luo et al., 2011,Cell 147: 409-422)

indicates data missing or illegible when filed

Analytical Ultracentrifugation-Sedimentation (SV) Experiments

Mixtures were loaded into SV chambers and equilibrated at 20° C. for 1 hbefore beginning the experiment. The sedimentation of the RIG-I:hairpincomplexes was monitored by absorbance at 260 nm, and the protein withoutRNA was monitored by absorbance at 280 nm.

Full length RIG-I protein was mixed with 5′ppp10L, 5′ppp20L, 5′ppp30Land 5′pppGC22 RNAs at a ratio of 4.5 μM RIG-I: 1.5 μM RNA (or RIG-Ialone) in 450 μl aliquots in buffer containing 25 mM HEPES pH 7.4, 150mM NaCl, 2 mM MgCl2, and 5 mM BME. The SV experiments were run at 40,000rpm in a Beckman Optima XL-I analytical ultracentrifuge. Partialspecific volumes for RIG-I and RIG-I:RNA complexes and buffer densityand viscosity parameters were calculated in SEDNTERP. Data analyses wereperformed in SEDFIT (Schuck, 2000, Biophysical Journal, 78: 1609-1619;Schuck et al, 2002, Biophysical Journal, 82: 1096-1111).

NADH-Coupled ATPase Experiments

ATPase activity of RIG-I was measured with the NADH-coupled ATPase assayadapted from previously described protocols (Luo et al., 2011, Cell147:409-422). Experiments were set up in 50 μl reaction volumes in 96well format using Corning clear half-area flat bottom plates (#3695).Each 50 μl reaction contained 10 μl of 5x NADH enzyme buffer (1 mM NADH,100 U of lactate dehydrogenase/mL, 500 U/mL of pyruvate kinase/mL, and2.5 mM phosphoenolpyruvate), 5-10 nM of RIG-I, 5 μl of varying amountsof either RNA or ATP, and a remaining volume of 25 mM3-(N-morpholino)propanesulfonic acid (MOPS) pH 7.4, 150 mM KCl, 2 mMDTT, and 0.1% Triton X-100. The rate of ATP hydrolysis was indirectlydetermined by monitoring the loss of NADH by reading the absorbance at340 nm using a Biotek Synergy H1 plate reader. For both the K_(m,ATP)and the K_(m,RNA) experiments, RIG-I and the RNA constructs were allowedto equilibrate for at least 2 hours before addition of ATP. Detergentwas required to record reproducible ATPase rates in the 96-well Corningclear bottom plates, especially at low concentrations of RIG-I. Theinitial velocities (ν0) at various RNA concentrations were plotted andfit to the following quadratic solution to the Briggs-Haldane equation:

$\begin{matrix}{y = {y_{0} + {({amp})*\frac{\left\lbrack M_{t} \right\rbrack + \left\lbrack S_{t} \right\rbrack + K_{m} - \sqrt{\left( {\left\lbrack M_{t} \right\rbrack + \left\lbrack S_{t} \right\rbrack + K_{m}} \right)^{2} - {{4\left\lbrack M_{t} \right\rbrack}\left\lbrack S_{t} \right\rbrack}}}{2\left\lbrack M_{t} \right\rbrack}}}} & (1)\end{matrix}$

[M_(t)] is the total protein concentration, [S_(t)] is the total [RNA],y₀ is 135 the basal activity, amp is the k_(cat) (minus the basalactivity), and Km is the apparent Michaelis constant for substrateactivation. The y₀ was constrained to the average basal activity fromthe entire set of 0 nM RNA, 5 mM ATP wells. The initial velocities (ν₀)at various ATP concentrations were plotted and fit to the hyperbolicform of the above equation:

$\begin{matrix}{y = \frac{({amp})*\left\lbrack {ATP} \right\rbrack}{K_{M} + \left\lbrack {ATP} \right\rbrack}} & (2)\end{matrix}$

For the ATPase experiments in which the ATP concentration was varied,the RNA concentration were held at 500 nM for the short RNA duplexes(FIGS. 4A-4D and FIGS. 7A-7C), 500 ng/μL for the poly I:C experiments(FIGS. 6A-6B), and 15 ng/μL for the poly I:C fractions (FIGS. 3A-3D).Although 15 ng/μL was suboptimal for the longer polylC fractions, it wasthe highest that could be managed for all of the fractions from a singlegel filtration experiment. One row from a 96 well plate constituted asingle experiment with the following 12 ATP solutions in μM (finalconcentrations listed) derived from a two-thirds dilution series: 0,30.2, 50.4, 84.0, 140.0, 233.3, 388.8, 648, 1080, 1800, 3000, and 5000.

For the ATPase experiments in which the RNA concentration was varied,the ATP concentration was held at 5 mM, approximately 10-fold above theK_(m,ATP) measured for each RNA fraction. One row from a 96-well plateconstituted a single experiment with the following 12 RNA concentrationsin nM (or ng/μl for poly I:C, final concentrations listed) from atwo-fold dilution series: 0, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.2, 62.5,125, 250, and 500 (FIGS. 4A-4D, FIGS. 6A-6B, and FIGS. 7A-7C). For thegel filtered poly I:C fractions the following 12 RNA concentrations in155 ng/μL (final concentrations listed) were used from a two-folddilution series: 0, 0.01, 0.03, 0.06, 0.12, 0.23, 0.47, 0.94, 1.88,3.75, 7.50, 15 (FIGS. 3A-3D). In order to calculate the nM amounts ofeach poly I:C fraction for FIG. 3D, the following estimates were madefor the duplex lengths of fractions A1-A7 respectively based on thesemi-denaturing polyacrylamide gel and molecular weight standards shownin FIG. 3A: 500, 360, 180, 90, 60, 40, and 25. The nM concentrationranges used for the analysis of the gel filtered poly I:C fractions isshown in Table 3.

Cell Culture IFN-β Response.

293T cells transfected with pUNO-RIG-I, pRL-TK and IFN-β/fireflyluciferase reporter were seeded at 15,000 cells per well, with each wellcontaining 5 μl of Lyovec (Invivogen) and either RNA hairpin or polyI:C. For luciferase measurements, the Promega Dual Luciferase Reporterassay system was used to quantify the cellular levels of firefly andRenilla luciferase.

Batches of 293T cells were grown to 70-80% confluency in 10 cm dishes inDulbecco's Modified Eagle Medium (DMEM; Invitrogen) containing 10%heat-inactivated fetal calf serum (Hyclone) and non-essential aminoacids (Invitrogen). For RIG-I transfections of 10 cm dishes of 293Tcells, one 800 μl aliquot of Opti-MEM containing 4 μg of pUNO-RIG-I, 1μg of pRL-TK, and 5 μg of an IFN-β/Firefly luciferase reporter plasmidwas mixed with a second 800 μl aliquot of Opti-MEM containing 50 μl oflipofectamine. After 45 minutes, the 1.6 mL aliquot was dilutedfour-fold with Opti-MEM and then added to a 10 cm dish of 293T cells.The transfection was allowed to proceed for 6-8 hours, and then 10 mL offresh DMEM was added to the plate. The cells were split twice into 15 cmdishes over the course of three days in 3 μg/μL blasticidin, and thenused for transfections in 96 well plates containing RNA hairpins or polyI:C fractions.

The 293T cells, in DMEM without blasticidin, and transfected withpUNO-RIG-I, pRL-TK, and IFN-β/Firefly luciferase reporter, were seededat 15,000 cells per well, with each well containing 5 μl of Lyovec(Invivogen), and the following final concentrations of 5′ppp RNA hairpinin nM: 39.1, 78.1, 156.3, 312.5, 625 or the following finalconcentrations of poly I:C in total ng per well: 15.6, 31.3, 62.5, 125,250, 500. In each experiment, the RNA hairpin or poly I:C was testedthree times at each concentration (or total RNA amount). Luminescencemeasurements were assayed between 16-24 hours after stimulation by theRNA.

For luciferase measurements, the Promega Dual Luciferase Reporter assaysystem was used to quantitate the cellular levels of firefly and Renillaluciferase. Briefly, media was aspirated from each 96 well plate andreplaced with 60 μL of passive lysis buffer. After 15 minutes at roomtemperature, lysates were collected, clarified by centrifugation, andthen 20 μl of lysate was assayed for firefly and Renilla luciferaseusing the luminometer from a Biotek Synergy H1 plate reader with a dualinjector. The Renilla luciferase is an internal control for eachexperiment and set of transfections, and the ratio of firefly luciferaseover Renilla luciferase is reported herein.

Accession Code

The atomic coordinates and structure factors of the binary complex ofRIG-I 191 (ΔCARDs: 1-229): 5′ OH-GC10 and the ternary complex of RIG-I(ΔCARDs: 1-229): 5′ OH-GC10: ADP-Mg′ have been deposited with the RCSBProtein Data Bank under the accession codes 3zd6 and 3zd7. The ternarycomplex of RIG-I (ΔCARDs:1-229): 5′OH-GC10: SO₄ ²⁺ is already availableunder the accession code 2ykg and has been previously published (Luo etal., 2011, Cell 147:409-422).

The results of the experiments are now described.

Further description of the data presented herein may be found in Kohlwayet al., 2013, EMBO Rep, 14(9): 772-9, the contents of which areincorporated herein by reference in its entirety.

HEL2i Movements Contribute to dsRNA Recognition

To understand the conformational changes that RIG-I undergoes during RNArecognition and surveillance, the conformations of RIG-I (ΔCARDs: 1-229)was visualized in complex with 5′OH-GC10 (FIG. 1A, Table 1), which showwell-ordered scanning movements of the HEL2i domain along the duplex RNAbackbone. Conformation 1 is the binary complex of RIG-I (ΔCARDs:1-229):5′OH-GC10, in which the ATP-binding pocket is empty and HEL2i stays inthe most compact state (FIG. 1A; pdb:3zd6). Conformation 2 is thepreviously reported crystal structure and is the ternary complex ofRIG-I (ΔCARDs:1-229): 5′OH-GC10:SO₄ ²⁻, in which the sulphate ionoccupies the ATP-binding pocket and HEL2i adopts an intermediate state(FIG. 1A, pdb:2ykg) (Luo et al., 2011, Cell 147:409-422). Conformation 3is also a ternary complex of RIG-I (ΔCARDs:1-229): 5′OH-GC10: ADP-Mg“,in which ADP-Mg” occupies the ATP-binding pocket and HEL2i adopts themost extended state (FIG. 1A; pdb:3zd7).

An alignment of the three RIG-I:RNA structures reveals that, while theHEL1-RNA-CTD forms a rigid sandwich-like fold, the HEL2i domain of RIG-Iis flexible and makes sequential contacts with several base pairs alongthe RNA duplex. Specifically, the HEL2i domain scans along the duplexbackbone between bases four through six of the 3′ bottom strand (thatis, the ‘tracking strand’ for SF2 helicase proteins) when transitioningbetween conformations, and then makes contact with the top strand in theextended, ADP-bound conformation (FIG. 1B). Two residues of the HEL2idomain, K508 and Q511, engage the RNA duplex: Q511 does not formcontacts with the RNA backbone in conformation 1; in conformation 2,Q511 interacts with the 2′-OH group of the fifth base of the bottomstrand; in conformation 3, Q511 reaches the 2′OH group of the fourthbase as the HEL2i domain slides along one face of the RNA duplex. K508comes into close contact with the RNA only in the extended conformation3, forming a salt bridge with the phosphate at position 9 on the 5′ topstrand (FIG. 1E).

During scanning, the pincer domain facilitates coordinated motion ofHEL2-HEL2i relative to HEL1 by engaging in a swinging motion along themore N-terminal α-helix whereas the C-terminal arm of the pincer servesas an anchor by remaining rigidly stacked against HEL1 (FIG. 1C). Subtlechanges in the ATP-binding pocket among the conformations are alsoobserved, including movements of the phosphate-binding loop and K270 ofmotif I, suggesting that the pincer and HEL2i motions might be linked toATP-binding and hydrolysis (FIG. 1D) (Luo et al., 2012b, Structure,20:1983-1988). Collectively, these conformations show dynamic openingand closing motions of the HEL2i domain along a 10 base pair stretch ofRNA. This led to the further investigation of two questions: (1) Howimportant are more RNA pairings that extend beyond this central core of10 base pairs at the helical terminus? That is, do more base pairscontribute to duplex RNA binding, stimulation of in vitro ATPaseactivity, or RIG-I-mediated IFN production? (2) How many RIG-I moleculesare necessary per RNA molecule to activate both ATPase activity and anIFN response?

RIG-I Binds Duplex RNA Termini as a Monomer

To study RIG-I binding to the internal duplex RNA regions, a family ofstructurally well-defined RNA hairpins was synthesized in which theduplex length was varied, but one terminus was blocked by the presenceof a structured, RNA tetraloop. A hydrodynamic method, sedimentationvelocity (SV), was employed to monitor populations of RIG-I andRIG-I:RNA complexes that form in the solution using hairpin duplexes of10, 20 and 30 base pairs in length, each bearing a single5′triphosphorylated end (5′ppp10L, 5′ppp20L, and 5′ppp30L, Table 2). Inaddition, we examined RIG-I binding to a 22mer duplex RNA that containstwo 5′triphosphorylated ends (5′pppGC22). It was observed that, atmicromolar concentrations of protein and RNA, RIG-I formed 1:1 complexeswith each hairpin tested, regardless of duplex length (FIG. 2).Specifically, peak s_(20,w) (standardized to 20° C. and water) values of6.0 for RIG-I alone, and 6.2, 6.4 and 6.9 for excess RIG-I with hairpinsof lengths 10, 20 and 30, respectively, were determined. By contrast,the complex of RIG-I with 5′pppGC22 had a s_(20,w) of 9.3, indicating a2:1 protein:RNA stoichiometry.

Kowalinksi et al (Kowalinski et al., 2011, Cell 147:423-435) alsodemonstrated that RIG-I binds with a 2:1 stoichiometry to a longer dsRNAthat has two blunt termini (61mer). This is consistent with the presentSV analysis, and taken together, these results show that RIG-Ispecifically recognizes the base-paired terminus of duplex RNA, and thatRIG-I does not form protein-protein-mediated oligomers even in thepresence of RNA (and ADP/ATP analogs, as shown in Luo et al (Luo et al.,2012b, Structure, 20:1983-1988)). Internal binding within the duplex isneither strongly favorable nor required for strong monomeric binding atthe 5′ end.

Table 2 Nucleic acid molecules used Name Sequence and Chemical Composition GC8 5′OH-GCGCGCGC-3′ (SEQ ID NO: 1) GC10 5′OH-GCGCGCGCGC-3′(SEQ ID ID NO: 2) GC12 5′OH-GCGCGCGCGCGC-3′ (SEQ ID NO: 3) GC145′OH-GCGCGCGCGCGCGC-3′ (SEQ ID NO: 4) GC18 5′OH-GCGCGCGCGCGCGCGCGC-3′(SEQ ID NO: 5) GC22 5′OHGCGCGCGCGCGCGCGCGCGCG C-3′ (SEQ ID NO: 6)5′pppGC10 5′ppp-GGCGCGCGCC-3′ (SEQ ID NO: 7) 5′pppGC125′ppp-GGCGCGCGCGCC-3′ (SEQ ID NO: 8) 5′pppCM12 5′ppp-GGACGUACGUCC-3′(SEQ ID NO: 9) 5′pppGC22 5′ppp-GGCGCGCGCGCGCG CGCGCGCC-3′(SEQ ID NO: 10) 5*ppp8L 5′ppp-GGCGCGGCUUCGGCCGCG  CC-3′(SEQ ID NO: 11)5′ppp10L 5′ppp-GGACGUACGUUUCGACGUAC GUCC-3′ (SEQ ID NO: 12) 5′ppp20L5′pppGGAUCGAUCGAUCGAUCGG CUUCGGCCGAUCGAUCGAU CGAUCC-3′(SEQ ID NO: 13)5*ppp30L 5′pppGGAUCGAUCGAUCGAUCGG CAUCGAUCGGCUUCGGCCGAUCGAUGCCGAUCGAUCGAUCGAUCC-3′  (SEQ ID NO: 14) polyI:C 5′OH-I^(n):C^(n)-3′(0.02-1 kilo base pairs)

RIG-I ATPase Activity is Dependent on Poly I:C Ends

To calibrate the present findings with those in the literature,RIG-I:RNA interactions were examined using a polymer that is moretypically used in studies of RIG-I. Specifically, RNA-stimulated ATPaseactivity was analyzed using poly I:C, which is a synthetic analogue ofdouble-stranded RNA that is commonly used for experimental stimulationof an IFN response. The ATPase activity of RIG-I is strictly dependenton the concentration of RNA, therefore the enzymatic activity of RIG-Ican be used as a metric for productive binding to poly I:C, or any otherRNA polymer. Poly I:C is a mixture of lengths and RNA conformationalstates, and it was thus hypothesized that RIG-I ATPase activity will bemore efficiently stimulated by shorter poly I:C fragments because theyhave more accessible ends per base pair. To test this hypothesis, ananalysis of RIG-I ATPase stimulation by low-molecular weight (LMW) polyI:C (FIGS. 6A-6B), which is a mixture of ˜25-500 base pair fragments,was conducted. To reduce heterogeneity of the poly I:C sample, the polyI:C was fractionated on an analytical Superdex 200 column to createseven fractions of decreasing size (FIG. 3A). The mean length of eachfraction was estimated, making the assumption that each fraction was adiscrete size, and thereby converted between ng/μl and nanomolar amountsof poly I:C strands (Table 3). Individual fractions were tested for theability to stimulate RIG-I ATPase activity by varying the poly I:Cfraction concentration at 5 mM ATP (K_(m,RNA), FIG. 3B) or by varyingthe ATP concentration at 15 ng/μl poly I:C fraction (K_(m,ATP), FIG.3C). Remarkably, a clear trend was found, demonstrating that shorterpoly I:C fragments stimulated RIG-I ATPase activity more effectively.The K_(m,RNA) for every fraction was plotted in terms of both ng/μl andnM poly I:C strands (FIG. 3D). Whereas the K_(m,RNA) spanned a 10-foldrange when expressed in ng/μl, the K_(m,RNA) varied approximatelytwo-fold or less when expressed in molarity of poly I:C strands. Infact, an identical K_(m,RNA) value of 20 nM was observed for bothfractions A1 and A7, which are at two extremes in terms of length, andthe K_(m,RNA) values for the other fractions were similar to this,within error. Furthermore, K_(m,ATP) values for each fraction of polyI:C (at saturating number of ends) were between ˜600 and 700 μM ATP(FIG. 3C). These data demonstrate that RIG-I ATPase activity isdependent on the number of duplex ends that are available in each polyI:C fraction, and they corroborate the view that internal duplex regionsare not critical for the enzymatic function of RIG-I.

TABLE 3 Poly I:C ng/μl to nanomolar concentrations. The estimates forthe length of each fraction of poly I:C as well as the approximatemolecular weights (FIG. 3A). The ng/μl concentrations used in the polyI:C K_(m, RNA) experiment were converted to nanomolar of poly I:Cstrands based on the estimated length and molecular weights of each polyI:C strand. Poly I:C (ng/μL) A1 (nM) A2 (nM) A3 (nM) A4 (nM) A5 (nM) A6(nM) A7 (nM) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.04 0.06 0.120.24 0.36 0.55 0.87 0.03 0.09 0.12 0.24 0.48 0.73 1.09 1.75 0.06 0.170.24 0.48 0.97 1.45 2.18 3.49 0.12 0.35 0.48 0.97 1.94 2.91 4.36 6.980.23 0.70 0.97 1.94 3.88 5.82 8.73 13.96 0.47 1.40 1.94 3.88 7.76 11.6417.45 27.93 0.94 2.79 3.88 7.76 15.51 23.27 34.91 55.85 1.88 5.59 7.7615.51 31.03 46.54 69.82 111.71 3.75 11.17 15.51 31.03 62.06 93.09 139.63223.41 7.50 22.34 31.03 62.06 124.12 186.18 279.26 446.82 15.00 44.6862.06 124.12 248.24 372.35 558.53 893.65 Length 500 360 180 90 60 40 25MW 335763 241706.16 120853.08 60426.54 40284.36 26856.24 16785.15

The Minimal RNA for Stimulating RIG-I ATPase Activity

To more precisely define the minimal duplex length for enzymaticactivation of RIG-I, the steady-state kinetic parameters for RIG-Iactivation by RNA hairpins and double-stranded duplexes ranging in sizefrom 8 to 30 base pairs, was measured with and without a 5′triphosphate(RNAs listed in Table 2). In order to evaluate the respective roles ofATP and RNA in activation of the RIG-I:RNA complex, the Michaelisconstant for ATP (K_(m,ATP)) at saturating RNA (500 nM) and theMichaelis constant for RNA (K_(m,RNA)) at saturating ATP (5 mM) wasmeasured for each RNA construct (k_(cat), K_(m,ATP) and K_(m,RNA)summary in Table 4).

Four 5′triphosphorylated RNA hairpins with duplex lengths of 8, 10, 20and 30 base pairs were tested for stimulation of RIG-I ATPase activity(FIGS. 4A-4D and FIGS. 7A-7C). The 5′ppp8L and 5′ppp10L hairpinsdisplayed the largest disparity in k_(cat), doubling from 7.45 s⁻¹ to14.32 s⁻¹ (ATP molecules hydrolysed per second per molecule of RIG-I) onthe addition of only two base pairs. The two larger constructs, 5′ppp20Land 5′ppp30L, were slightly less effective at stimulation than 5′ppp10L,with k_(cat) values of 12.59 s⁻¹ and 9.98 s⁻¹, respectively. It isinteresting that, the 5′ppp8L hairpin stimulated RIG-I ATPase activityto a lesser degree than 5′ppp10L. This result is intriguing in thecontext of the structural data, as it provides further support for thehypothesis that two extra base pairs beyond the footprint of RIG-I, asin the 5′ppp10L hairpin, likely provide the HEL2i domain with therequired room for full flexibility and the coordinated internal motionsthat lead to efficient ATP hydrolysis.

In order to more comprehensively evaluate the RNA length dependence forRIG-I ligands, six ‘GC’ palindromic, blunt-ended, 5′hydroxyl RNAduplexes with lengths of 8, 10, 12, 14, 18 and 22 were tested forstimulation of RIG-I ATPase activity (FIGS. 4A-4D and FIGS. 7A-7C).Remarkably, the k_(cat) values at saturating ATP and RNA concentrationsfollowed the same trends as the 5′triphosphorylated hairpins. The peakATPase activity occurred with stimulation from GC12, albeit withnegligible differences in comparison to stimulation from GC10 or GC14.Taken together, the length dependence of the k_(cat) for thepalindromic, 5′hydroxyl duplexes were qualitatively similar to the5′triphosphorylated hairpins. In each case, robust ATPase activity forRIG-I stimulation was observed with RNA of at least 10 base pairs inlength, regardless of the presence of a 5′-triphosphate moiety, andactivity declined slightly with increasing duplex length.

TABLE 4 RNA stimulated ATP hydrolysis by RIG-I. k_(cat) ± SD K_(m, ATP)± SD K_(m, RNA) ± SD RNA construct (s⁻¹RIG-I⁻¹) (μM) (nM) 5′ppp8L  7.45± 0.90 454 ± 18  5.16 ± 0.40 5′ppp10L 14.3 ± 2.5 556 ± 65  4.46 ± 0.505′ppp20L 12.6 ± 1.6 604 ± 40  10.1 ± 0.50 5′ppp30L 9.98 ± 1.3  622 ± 10010.8 ± 1.0 GC8 7.34 ± 2.6 425 ± 66 91.4 ± 15  GC10 14.4 ± 3.0 511 ± 5524.4 ± 1.5 GC12 15.9 ± 3.1 528 ± 38 13.1 ± 1.0 GC14 15.1 ± 1.9 537 ± 3123.2 ± 1.3 GC18 12.5 ± 1.3 600 ± 37 26.7 ± 3.4 GC22 11.3 ± 1.1 570 ± 7527.3 ± 1.1 5′pppGC10 18.8 ± 2.8 498 ± 35  1.16 ± 0.20 5′pppGC12 20.5 ±3.3 535 ± 55  2.33 ± 0.20 5′pppCM12 15.9 ± 1.8 591 ± 57  2.62 ± 0.105′pppGC22 12.3 ± 1.9 536 ± 39 3.58 ± 1.0 LMW poly I:C  4.90 ± 0.50  690± 130    2.40 ± 1.1 (ng/μl) ***Note that for the poly I:C K_(m, ATP),the poly I:C concentration was kept at 500 ng/μl. For the poly I:CK_(m, RNA), the poly I:C concentration was varied up to 500 ng/μl.

5′Ppp Enhances RNA Binding, but not ATP Hydrolysis

The trends in k_(cat) values for the 5′triphosphorylated hairpins andthe 5′hydroxyl duplexes were similar despite tighter RNA binding(reflected by smaller K_(m,RNA) values) by the 5′triphosphorylatedhairpins. This finding reveals that the 5′triphosphate might functionprimarily at the step of binding and that it does not have a majorimpact on ATP hydrolysis. To further investigate the function of thetriphosphate, three ‘GC’ palindromic blunt-ended RNA duplexes with5′triphosphates of lengths 10, 12 and 22 were tested for stimulation ofRIG-I ATPase activity, as well as a 5′triphosphorylated 12-mer,5′pppCM12, containing a palindromic but non-uniform sequence includingall four nucleotides (FIGS. 4A-4D and FIGS. 7A-7C). Although the k_(cat)for 5′pppGC10 and 5′pppGC12 were marginally higher than GC10 and GC12,the measured k_(cat) for 5′pppCM12 was identical to GC12, and thek_(cat) for 5′pppGC22 was within the experimental error of the k_(cat)for GC22. These data further demonstrate that the triphosphate has aminimal effect on the k_(cat) values for RIG-I ATP hydrolysis when allligands are saturating.

Only small changes in the measured K_(m,ATP) (the apparent bindingconstant for ATP) was observed for the RNA constructs tested (FIG. 4A).Specifically, the K_(m,ATP) were between ˜500 and 600 μM, implying thatthe conformational changes in RIG-I that are required for catalysis arenot influenced by the presence of a triphosphate moiety or duplexlength. This observation is corroborated in part by structural evidenceshowing that RIG-I binds 5′triphosphorylated RNA identically to5′hydroxyl RNA (Luo et al., 2012b, Structure, 20:1983-1988).

By contrast, the K_(m,RNA) (the apparent binding constant for RNA)directly correlated with the presence of a 5′triphosphate on the RNAhairpin or duplex (FIG. 4B). The K_(m,RNA) for the four5′triphosphorylated duplexes were between 1.2 and 3.6 nM, and theK_(m,RNA) for the four 5′triphosphorylated hairpins were between 5.2 and10.8 nM. However, the 5′hydroxyl RNA duplexes yielded K_(m,RNA) valuesbetween 20 and 30 nM, with the exception of GC8 (91 nM) and GC12 (13nM). These data underscore the fact that any RNA duplex of theappropriate length (>10 bp) can fully stimulate the ATPase activity ofRIG-I, but a duplex containing a 5′-triphosphate binds RIG-I with higheraffinity and will therefore stimulate ATPase activity at lower RNAconcentrations. This finding is an important distinction that explainswhy only trace amounts of viral RNA might be required to activate theinterferon-0 (IFN-(3) response in infected cells. Interestingly, thepoly I:C fractions exhibited a similar range of K_(m,RNA) values asthose observed for the 5′hydroxyl duplex RNA, suggesting that RIG-Ifunctions on poly I:C in a manner that is similar to any other RNAduplex that lacks a 5′-triphosphate.

RIG-I ATPase Activity on a Monomeric and Dimeric RNA Ligands Suggests NoFunctional Intermolecular Interactions Between RIG-I

Having identified a series of important intramolecular dynamics thatcontribute to RIG-I function, the next set of experiments was designedto determine whether functional intermolecular interactions betweenRIG-I molecules might also play a role in in establishing acatalytically competent ternary complex. The ATPase activity of RIG-Iwas therefore measured at protein concentrations varying between 5 and50 nM using saturating concentrations of 5′ppp10L hairpin and 5′pppGC22duplex. Without wishing to be bound by any particular theory, it isbelieved that if the ATPase activity of RIG-I is modulated by homotypicprotein:protein interactions, then the catalytic activity of the proteinwould be expected to exhibit a non-linear relationship with enzymeconcentration.

The k_(CAT) for RIG-I measured on both 5′ppp10L and 5′pppGC22 does notvary within the ten-fold range of RIG-I concentrations tested (FIG. 10A,FIG. 10B). Additionally, there is an approximate 25% decrease in themeasured k_(CAT) between the monomeric RIG-I substrate, 5′ppp10L, andthe dimeric RIG-I substrate, 5′pppGC22 (FIG. 10C). The lack of asignificant change in the k_(CAT), either from an increase in enzymeconcentration or from potential 5′pppGC22-induced oligomerizationindicates that RIG-I functions optimally as a monomer. Theseobservations indicate that protein-protein interactions do not alter theability of RIG-I to hydrolyze ATP, either on the same RNA molecule, aswith 5′pppGC22, or between RIG-I:RNA complexes.

1:1 RIG-I:RNA Binding is Sufficient to Stimulate IFN-β

The in vitro SV and RNA-stimulated ATPase studies provide strongevidence that RIG-I activation requires only the 5′ terminus of duplexRNA, along with an adjacent 10-12 base pairs. And while RIG-I in vitroactivity is typically associated with RNA binding or ATPase activity,the direct relationship to interferon stimulation is not clear.Therefore, it was important to test the relevance of the present invitro results in cell culture. To accomplish this, the ability of5′triphosphorylated hairpins and poly I:C fractions to stimulate aRIG-I-mediated IFN-β response in 293T cells was measured (FIG. 4D).

Remarkably, it was found that three of the four hairpins—5′ppp10L,5′ppp20L and 5′ppp30L—stimulated an IFN-β response comparable to thepositive controls, LMW poly I:C and 5′pppGC22 (mock control in FIG. 8).Both LMW poly I:C and short 19 bp+ RNA duplexes have been shown to begood activators of RIG-I (Kato et al., J Exp Med, 205, 1601-1610; Schleeet al., 2009, Immunity, 31: 25-34).

Further, it is demonstrated that the “GC” palindromic RNAs alsostimulate an IFN-β response (FIG. 11). While in certain instances thepalindromic RNAs do not exhibit an IFN-β response to the same level asthe hairpins, the present data demonstrates that they may be used topromote IFN-β production. While not wishing to be bound by anyparticular theory, the hairpins may be a superior stimulant for RIG-Isimply because of the ability to re-anneal after being unwound, whereasthe shorter palindromic duplexes would likely lose their ability tostimulate RIG-I as soon as the duplex melted.

The IFN-β stimulation from the 5′ppp10L construct is of particularinterest because it strongly supports the idea that RIG-I does notsurvey the cell as an oligomer, that RIG-I does not need to oligomerizeon a target RNA duplex strand to elicit an IFN-β response, and thatRIG-I does not need to translocate on duplex RNA regions to elicit anIFN-β response. Consistent with these findings, even the shortest polyI:C fragments fully stimulated the IFN response in cells (FIG. 9). Theslightly better IFN production, especially at lower RNA concentrations,for 5′ppp20L and 5′ppp30L, can be attributed to the fact that they aremore stable duplexes that likely have a longer half-life in the cell.

Model for RNA Surveillance by RIG-I

Recent structural studies have shed new light on RNA surveillance byRIG-I. In all cases, RIG-I is shown to bind RNA molecules as a monomerand to interact specifically with the terminus of an RNA duplex. Indeed,it has been called an end-capper (Kowalinski et al., 2011, Cell, 147:423-435). Intriguingly, RIG-I is observed to bind all blunt RNA terminiin much the same way, without regard to RNA sequence or the presence ofa 5′-triphosphate (Jiang et al., 2011, Nature, 479: 423-427; Luo et al.,2011, Cell, 147: 409-422). While these crystallographic observations areuseful, they do not establish the minimal length of RIG-I PAMPs insolution for binding, for ATPase activity, and ultimately for signallingin the cell. In addition, the issue of cooperative RIG-I multimerizationon RNA has not been squarely addressed. Given the importance of theseissues, and of RIG-I activation in general, it was decided to use acombination of techniques to define the minimal RNA PAMP that isrequired for full activation of RIG-I in vitro and in mammalian cells.

The findings presented herein indicate that the minimal RNA PAMP that isrequired for activation in vitro and in cell culture has been defined,and that determinants under all conditions agree: the RIG-I monomer isactivated upon binding the blunt terminus of a RNA duplex. The proteininteracts with the 10 base pairs adjacent to the 5′ end with an affinitythat is enhanced by the presence of a 5′triphosphate. Collectively, theavailable data in the literature suggest that these 1:1 RIG-I:RNA(end)complexes might then oligomerize into higher order complexes via theCARD domains, resulting in a model that is consistent with findings ondownstream events that have been reported by others (FIGS. 5A-5B) (Jianget al., 2012, Immunity, 36: 959-973; Gack et al., 2007, Nature, 446:916-920). As presented herein, the minimal determinants for functionalRNA recognition by RIG-I is identified. Further, it is demonstrated thatRIG-I uses its functional domains collaboratively to accomplish specificantiviral surveillance in a complex intracellular environment.

Example 2: Ability of Small Hairpin RNAs to Induce Interferon In Vivo

Experiments were conducted to examine the ability of small hairpin RNAsto induce interferon production in vivo. Mice were injected in the tailvein with jetPEI/RNA complex (i.v.), and serum was collected at 5 hourspost-injection. The dose used per mouse was as follows: polyIC=25ug,hp10=640 uM (25.15ug), hp414=640 uM (33.4ug). 4 mice were used for eachcondition. Blood was collected five hours post-injection. The blood wasleft at 4° overnight to clot. It was then centrifuged for 30 minutes at4° (3000 prm), and the serum (supernatant) was collected. The resultsindicate that very high levels of IFNalpha are induced by shRNAs andpolyIC, and not by the vehicle control (FIG. 12). Notably, the shRNAsinduce more IFNalpha than polyIC. Note that hp10 is a5′-triphosphorylated 10 base-pair duplex with a UUCG tetraloop at oneend (same as 5′ppp10L from FIGS. 4A-4D and FIGS. 7A-7C) and hp14 is a5′-triphosphorylated 14 base pair duplex with a UUCG tetraloop at oneend. The polyIC is low molecular weight poly IC.

Further experiments were conducted to compare IFNα production induced bythree different RNA constructs. Mice were injected in the tail vein withjetPEI/RNA complex (i.v.), and serum was collected at 5 hourspost-injection, n=3 per group. The first construct is 5′ppp10Ltranscribed and treated with Dnase/Prot K, purified using phenolextraction and EtOH precipitation. The second construct is 5′OH10L,which is the 5′ppp10L, treated and purified as above, and then treatedwith CIP. The third construct is a synthesized and abological form of5′ppp10L. Blood was collected five hours post-injection. The blood wasleft at 4° overnight to clot. It was then centrifuged for 30 minutes at4° (3000 prm), and the serum (supernatant) was collected. It wasobserved that only 5′ppp10L (whether transcribed or synthetic), and notRNA lacking triphosphate, induces interferon (FIG. 13). Both transcribedand synthesized 5′ppp10L induce IFN to a similar degree, although thesynthetic triphosphorylated RNA is slightly more active. Extra enzymetreatment and purification of transcribed 5′ppp10L does not impact IFNlevels (as compared to data shown in FIG. 12).

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1-11. (canceled)
 12. A method for inducing type I interferon productionin a cell, the method comprising contacting the cell with a nucleic acidmolecule, wherein the molecule comprises a double-stranded section ofless than 19 base pairs and at least one blunt end.
 13. The method ofclaim 12, wherein the nucleic acid molecule comprises a single chainmolecule and forms a hairpin structure comprising the double-strandedsection and a loop.
 14. The method of claim 12, wherein the nucleic acidmolecule comprises a double chain molecule and two blunt ends.
 15. Themethod of claim 12, wherein the nucleic acid molecule comprises at leastone of the group consisting of a 5′ triphosphate and a 5′ diphosphate.16. The method of claim 12, wherein the molecule is capable of enteringthe nucleus.
 17. The method of claim 12, wherein the molecule comprisesa modified phosphodiester backbone.
 18. The method of claim 12, whereinthe molecule comprises at least one 2′-modified nucleotide.
 19. Themethod of claim 18, wherein the 2′-modified nucleotide comprises amodification selected from the group consisting of: 2′-deoxy,2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), and 2′-O—N-methylacetamido (2′-O-NMA).
 20. The method ofclaim 12, wherein the molecule comprises at least one modified phosphategroup.
 21. The method of claim 12, wherein the molecule comprises atleast one modified base.
 22. The method of claim 12, wherein thedouble-stranded section comprises one or more mispaired bases.
 23. Amethod for treating a disease or disorder in a subject, wherein thedisease or disorder is selected from the group consisting of a bacterialinfection, a viral infection, a parasitic infection, cancer, anautoimmune disease, an inflammatory disorder, and a respiratorydisorder, the method comprising administering to the subject atherapeutically effective amount of a nucleic acid molecule, wherein themolecule comprises a double-stranded section of less than 19 base pairsand at least one blunt end, and wherein the administering induces type Iinterferon production in at least one cell of the subject.
 24. Themethod of claim 23, wherein the nucleic acid molecule comprises a singlechain molecule and forms a hairpin structure comprising thedouble-stranded section and a loop.
 25. The method of claim 23, whereinthe nucleic acid molecule comprises a double chain molecule and twoblunt ends.
 26. The method of claim 23, wherein the nucleic acidmolecule comprises at least one of the group consisting of a 5′triphosphate and a 5′ diphosphate.
 27. The method of claim 23, whereinthe molecule is capable of entering the nucleus.
 28. The method of claim23, wherein the molecule comprises a modified phosphodiester backbone.29. The method of claim 23, wherein the molecule comprises at least one2′-modified nucleotide.
 30. The method of claim 29, wherein the2′-modified nucleotide comprises a modification selected from the groupconsisting of: 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and2′-O—N-methylacetamido (2′-O-NMA).
 31. The method of claim 23, whereinthe molecule comprises at least one modified phosphate group.
 32. Themethod of claim 23, wherein the molecule comprises at least one modifiedbase.
 33. The method of claim 19, wherein the double-stranded sectioncomprises one or more mispaired bases.
 34. The method of claim 23,wherein the molecule comprises a loop region. 35.-40. (canceled)